Photosensitizer conjugates for pathogen targeting

ABSTRACT

Conjugate molecules which include photosensitizer compositions conjugated to non-antibody non-affinity pair targeting moieties and methods of making and using such conjugates are described.

GOVERNMENT FUNDING

This invention was made with government support from the NationalInstitutes of Health grant NIH RO1 AR40352 and from the Office of NavalResearch. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to a conjugate which includes a photosensitizerand a targeting moiety, and methods of using the conjugate.

BACKGROUND OF THE INVENTION

Infectious diseases remain an unsolved problem, due largely to emergenceof multiply-antibiotic resistant strains of bacteria, newly discoveredviral diseases, and the spread of fungal and protozoan diseases.

Advanced periodontal disease is one of a large number of oral infectiousdiseases, and is the principal cause of tooth loss in those over 30years old. Periodontal diseases arise from the interaction betweenbacterial cells and their products in dental plaque, and the hostdefense mechanisms (Antczak-Bouckoms, A., (1994), J. Dent. Educ.58:625-640). Current treatments often rely on mechanical removal of theplaque and bacteria, which can be inefficient (Unsal E. et al., (1995),J. Periodontol. 66:47-51), or antibiotic therapy, which can lead tobacterial resistance (Olsvik, B. et al., (1995), J. Clin. Periodontol.22:391-396).

Photodynamic therapy (PDT) has been proposed as an attractive method ofeliminating oral bacteria and bacteria in topical and gastrointestinalinfections because these sites are relatively accessible toillumination. For example, fiber optics can be used to deliver lightinto the dental pocket (Wilson, M., (1993), J. Appl. Bacteriol.75:299-306).

SUMMARY OF THE INVENTION

The inventor has discovered that classes of molecules not hither to usedas targeting moieties for photosensitizers, can be used to targetphotosensitizers.

Accordingly, the invention features, a conjugate molecule which includesa photosensitizer coupled to a non-pair member (NPM) moiety, e.g., anNPM-polypeptide.

In embodiments in which the targeting moiety includes a polypeptide, thetargeting moiety can be a linear, branched, or cyclic polypeptide.

In preferred embodiments, the targeting moiety includes a smallanti-microbial peptide (SAMP). Histatins, defensins, cecropins,magainins, Gram positive bacteriocins, and peptide antibiotics can beSAMP's. In preferred embodiments, the targeting moiety includes abacterial, fungal, animal, e.g., mammalian, e.g., human, SAMP, or anactive fragment or analog thereof.

In preferred embodiments the targeting moiety includes a defensin, or anactive fragment or analog thereof. By way of example the defensin canbe: a human defensin, e.g., HNP-1, -2, -3, or -4; a guinea pig defensin,e.g., GPNP; a rabbit defensin, e.g., rabbit NP-1, -2, -3A, -3B, or 5; arat defensin, e.g., rat NP-1, -2, -3, or -4; murine cryptin; bovinegranulocyte bactenecin or indolicidin; or bovine seminal plasmin.

In preferred embodiments, the targeting moiety includes a SAMP of insectorigin, or an active fragment or analog thereof, e.g., a cecropin fromCecropia moths, bumble bees, fruit flies, or other insects, an apidaecinfrom honeybees, or an adropin from fruit flies.

In preferred embodiments, the targeting moiety includes a SAMP ofamphibial origin, or an active fragment or analog thereof, e.g., amagainin, a PGLA, a XPF, a LPF, a CPG, a PGQ, a bombinin, abombinin-like peptide BLP-1, -2, -3, or -4, or a brevinin.

In preferred embodiments the targeting moiety includes a SAMP from aninvertebrate, or an active fragment, or analog thereof, e.g.,tachyplesin I, II, or III, or polyphemusin I or II, from horseshoe crab.In preferred embodiments, the targeting moiety is from a fish, e.g.,pardaxin.

In preferred embodiments, the targeting moiety includes a bacteriocin,more preferably a Gram positive bacteriocin, or an active fragment, oranalog thereof, e.g., a nisin, a subtilin, epidermin, gallidermin,salivarin, or a lacticin.

In preferred embodiments, the targeting moiety includes a peptideantibiotic, or an active fragment or analog thereof, e.g., a tyrocidin,or a bacitracin.

In preferred embodiments the targeting moiety includes a histatin, or anactive fragment or analog thereof, e.g., histatin-1 through -8,preferably histatin-1, -3, or -5. In preferred embodiments the targetingmoiety includes histatin-5 residues 13-24, or corresponding residuesfrom other histatins. In preferred embodiments the targeting moietyincludes a histatin molecule which has been engineered to include aninternal duplication.

In preferred embodiments, the targeting moiety includes a polypeptidehaving an affinity for a polysaccharide target, e.g., a lectin. By wayof example the lectin can be a seed, bean, root, bark, seaweed, fungal,bacteria, or invertebrate lectin. In preferred embodiments, thetargeting moiety includes a plant polypeptide, e.g., a lectin from jackbean, e.g., concanavalin A, or a lectin from a lentil, Lens culinaris.

In preferred embodiments, the targeting moiety includes a salivarypolypeptide, or an active fragment or analog thereof. Examples ofsalivary polypeptides are the histatins, e.g., histatin-1 through -8, ormore preferably, histatin-1, -3, or -5. In preferred embodiments thetargeting moiety includes histatin-5 residues 13-24, or correspondingresidues from other histatins. In preferred embodiments the targetingmoiety includes a histatin molecule which has been engineered to includean internal duplication.

In preferred embodiments, the targeting moiety includes a Gram negativebacteriocin, e.g., colicin B, colicin E1, or colicin Ia.

In preferred embodiments the targeting moiety includes bacteriallyelaborated polypeptide, e.g., nisin, subtilin, epidermin, gallidermin,salivarin, or lacticin.

In preferred embodiments the targeting moiety includes a molecule, e.g.,a peptide, other than an antibody or either member of a receptor-ligandpair.

In preferred embodiments, the conjugate does not include, e.g., it isnot coupled, e.g., covalently or non-covalently coupled to: a PM; anantibody; an enzyme; a hormone; a receptor on a cell surface; or theligand for a receptor on a cell surface.

In preferred embodiments the targeting moiety includes a peptide inwhich at least 10, 20, 30, 40, 50, 60, 70, 80, 90% of the amino acidresidues are of one amino acid residue, e.g., a positively charged aminoacid residue, e.g., a lysine reside, an arginine residue, or anornithine residue. Particularly preferred targeting moieties arepolyamino acids, e.g., polylysine, polyarginine, or polyornithine.

In preferred embodiments the targeting moiety: is cationic; has a netpositive charge of +1, +2 or +3 per molecule; has a net positive chargeequal to or greater than +4; includes a positively charged amino acidresidue, e.g, lysine; includes at least 2, 3, 4, or more positivelycharged amino acid residues, e.g, a lysine, arginine, or ornithineresidue.

In other embodiments the targeting moiety: is anionic; has a netnegative charge of -1, -2 or -3 per molecule; has a net negative chargeequal to or greater than -4; includes a negatively charged amino acidresidue, e.g, aspartic acid or glutamic acid; includes at least 2, 3, 4,or more negatively charged amino acid residues, e.g, glutamic; includesat least 10, 20, 30, 40, or 50% or more negatively charged amino acidresidues, e.g. aspartic acid, or glutamic acid.

In preferred embodiments the targeting moiety: is approximately neutralin charge; includes at least 50, 60, 70, 80, or 90% amino acid residueswhich are neutral amino acid residues, such as serine, threonine,alanine, methionine, cysteine, or valine.

In preferred embodiments the targeting moiety has a molecular weight ofmore than 1200, 1800, 2400, 3000, 6000, 10,000, 25,000, 50,000, 100,000,or 200,000 daltons. In preferred embodiments the targeting moiety has amolecular weight of less than 250,000, 150,000, 60,000, 25,000, 10,000,8,000, or 6,000 daltons. In particularly preferred embodiments themolecular weight of the targeting moiety is between 300 and 1800, 600and 2400, 1200 and 6,000, 5,000 and 8,000, 8,000 and 15,000, 15,000 and30,000, 35,000 and 70,000, 70,000 and 150,000, or 150,000 and 300,000daltons.

In preferred embodiments the targeting moiety includes a peptide atleast 3, 6, 12, 18, 24, 30, 60, 100, 250, 500, 1,000, or 2,500 residuesin length. In preferred embodiments the targeting moiety is a peptideless than 3,000, 1,500, 700, 300, 150, 100, 80, 60,40, 30, or 15residues in length. In particularly preferred embodiments the targetingmoiety includes a peptide of between 6 and 15, 12 and 18, 18 and 30, 20and 40, 30 and 60, 80 and 120, 150 and 300, 300 and 600, 800 and 1,200,or 2,000 and 3,000 residues in length.

In preferred embodiments the targeting moiety includes a protein whichforms a pore in the permeability barrier of the target organism, e.g.,in Staphylococcus aureus, Klebsiella pneumoniae, Candida albicans,Leishmania donovani, or Giardia lamblia.

In other preferred embodiments, the targeting moiety includes a lowdensity lipoprotein, a high density lipoprotein or a very low densitylipoprotein.

In preferred embodiments, the targeting moiety has been selected using asurface molecule of the target organism as an affinity selection orscreen, e,g, the targeting moiety has been selected in a chemical orphage display library.

In particularly preferred embodiments the targeting moiety includes apolylysine molecule. The polylysine can be between 6 and 15, 12 and 18,18 and 30, 20 and 40, 30 and 60, 80 and 120, 150 and 300, 300 and 600,800 and 1,200, or 2,000 and 3,000 residues in length.

In preferred embodiments the targeting moiety includes a polypeptide,e.g., a polyamino acid, which has been chemically modified to alter itscharge, e.g., the charge of side chains of one or more amino acidresidues of the polyamino acid. For example, one or more, orapproximately 10, 25, 50, 75, 90 or 100% of the charged side chains canbe modified. By modified is meant that a negative side chain, e.g., aglutamic acid, or an aspartic acid, side chain is made positive orneutral in charge, a positively charged side chain, e.g., the side chainof lysine, arginine, or ornithine is made negative or neutral in charge.By way of example, one or more of the side chains of polylysine can bemade neutral or negative in charge.

In preferred embodiments: the photosensitizer produces singlet oxygenupon absorption of electromagnetic irradiation at the proper energylevel and wavelength; the photosensitizer includes a porphyrin orporphyrin derivative; the photosensitizer includes chlorin e6 or achlorin derivative.

In preferred embodiments the conjugate further includes a backbonemember. In such embodiments the backbone is coupled both to aphotosensitizer and to a targeting moiety. The backbone can itself alsobe a targeting moiety, e.g. polylysine.

In preferred embodiments, the conjugate molecule has affinity for atarget organism. The target organism, by way of example, can be: amicroorganism, e.g., a bacterial cell, a fungal cell, a protozoan cell,a cell of Pneumocystis carinii; a virus; or, a parasitic helminth; or anarthropod.

In preferred embodiments where the cell is a bacterial cell, thebacterial cell can be a Staphylococcus, Streptococcus, Enterococcus,Mycobacterium, Pseudomonas, Salmonella, Shigella, Escherichia, Erwinia,Klebsiella, Borrelia, Treponema, Campylobacter, Helicobacter,Bordetella, Neisseria, Legionella, Leptospira, Serpulina, Mycoplasma,Bacteroides, Klebsiella, Yersinia, Chlamydia, Vibrio, Actinobacillus,Porphyria, Hemophilus, Pasteurella, Peptostreptococcus, Listeria,Propionibacterium, Mycobacterium, Corynebacterium or Dermatophilus cell.

In preferred embodiments where the cell is a fungal cell, the cell canbe a Candida or an Aspergillus cell.

In preferred embodiments, the organism is Pneumocystis carinii.

In preferred embodiments where the target organism is a protozoan cell,the cell is an Entamoeba, a Toxoplasma, a Giardia, a Leishmania, aCrytosporidium, or a Schistosoma.

In preferred embodiments where the target organism is a virus, the virusis an HIV, an HTLV, a hepatitis virus, an influenza virus, a rhinovirus,a papilloma virus, a measles virus, a Herpes virus, a rotavirus, aparvovirus, a psittacosis virus, or an Ebola virus.

In preferred embodiments where the target organism is an arthropod, thearthropod is a parasitic mite.

In preferred embodiments where the target organism is a helminth, thehelminth is a nematode or a trematode.

In preferred embodiments the target organism is an oral bacterialspecies, e.g., Porphyromonas (Bacteroides) gingivalis.

In another aspect, the invention features a conjugate molecule whichincludes a photosensitizer coupled to a non-pair member (NPM) targetingmoiety and a pharmaceutically acceptable carrier.

In another aspect, the invention features, a conjugate molecule whichincludes a photosensitizer coupled to a targeting moiety which includesa non-pair member (NPM) polypeptide moiety having affinity for an oralbacterial species.

In embodiments in which the targeting moiety includes a polypeptide, thetargeting moiety can be a linear, branched, or cyclic polypeptide.

In particularly preferred embodiments the targeting moiety includes apolylysine molecule. The polylysine can be between 6 and 15, 12 and 18,18 and 30, 20 and 40, 30 and 60, 80 and 120, 150 and 300, 300 and 600,800 and 1,200, or 2,000 and 3,000 residues in length.

In preferred embodiments the targeting moiety includes a polypeptide,e.g., a polyamino acid, which has been chemically modified to alter itscharge, e.g., the charge of side chains of one or more amino acidresidues of the polyamino acid. For example, one or more, orapproximately 10, 25, 50, 75, 90 or 100% of the charged side cains canbe modified. By modified is meant that a negative side chain, e.g., aglutamic acid, or an aspartic acid, side chain is made positive orneutral in charge, a positively charged side chain, e.g., the side chainof lysine, arginine, or ornithine is made negative or neutral in charge.By way of example, one or more of the side chains of polylysine can bemade neutral or negative in charge.

In preferred embodiments: the photosensitizer produces singlet oxygenupon absorption of electromagnetic irradiation at the proper energylevel and wavelength; the photosensitizer includes a porphyrin orporphyrin derivative; the photosensitizer includes chlorin e6 or achlorin derivative.

In preferred embodiments the conjugate further includes a backbonemember. In such embodiments the backbone is coupled both to aphotosensitizer and to a targeting moiety. The backbone can itself alsobe a targeting moiety, e.g. polylysine.

In preferred embodiments the conjugate includes chlorin e6 conjugated topolylysine, e.g., 1 or 2 to 20 chlorin e6 molecules conjugated to apolylysine between about 1,000 and 3,000 in molecular weight.

In preferred embodiments the conjugate includes chlorin e6 conjugated toa histatin polypeptide, or an active fragment or analog thereof, e.g., 1or 2 to 4 chlorin e6 molecules conjugated to a histatin-5 polypeptide.

In preferred embodiments the conjugate includes chlorin e6 and ahistatin polypeptide, or an active fragment or analog thereof,conjugated to a polylysine backbone, e.g., either from one to 4polylysine chains (MW 1,000 to 3,000 daltons, each containing from 1 or2 to 20 chlorin e6 molecules) joined to one histatin-5 polypeptide, orfrom one to 4 histatin-5 polypeptides joined to a polylysine chain (MW1,000 to 3,000 and containing 1 or 2 to 16 chlorin e6 molecules.

In preferred embodiments the target organism is an oral bacterialspecies, e.g., Porphyromonas (Bacteroides) gingivalis.

In preferred embodiments, the conjugate does not include, e.g., it isnot coupled, e.g., covalently or non-covalently coupled to: a PM; anantibody; an enzyme; a hormone; a receptor on a cell surface; or theligand for a receptor on a cell surface.

In preferred embodiments, the targeting moiety includes a salivarypolypeptide, or an active fragment or analog thereof. Examples ofsalivary polypeptides are the histatins, e.g., histatin-1 through -8, ormore preferably, histatin-1, -3, or -5. In preferred embodiments thetargeting moiety includes histatin-5 residues 13-24, or correspondingresidues from other histatins. In preferred embodiments the targetingmoiety includes a histatin molecule which has been engineered to includean internal duplication.

In another aspect, the invention features, a method of treating asubject, for a disorder characterized by the presence of an unwantedorganism. The method includes:

administering to the subject, a conjugate which includes aphotosensitizer coupled to a NPM targeting moiety, e.g., a conjugatedescribed herein;

irradiating the subject with energy of a wavelength appropriate toproduce a cytotoxic effect by the photosensitizer;

thereby treating the subject, for the disorder characterized by thepresence of an unwanted organism.

In preferred embodiments, the unwanted organism, by way of example, canbe: a microorganism, e.g., a bacterial cell, a fungal cell, a protozoancell, a cell of Pneumocystis carinii; a virus; or, a parasitic helminth;or an arthropod.

In preferred embodiments where the unwanted organism is a bacterialcell, the bacterial cell can be a Staphylococcus, Streptococcus,Enterococcus, Mycobacterium, Pseudomonas, Salmonella, Shigella,Escherichia, Erwinia, Klebsiella, Borrelia, Treponema, Campylobacter,Helicobacter, Bordetella, Neisseria, Legionella, Leptospira, Serpulina,Mycoplasma, Bacteroides, Klebsiella, Yersinia, Chlamydia, Vibrio,Actinobacillus, Porphyria, Hemophilus, Pasteurella, Peptostreptococcus,Listeria, Propionibacterium, Mycobacterium, Corynebacterium orDermatophilus cell. In more preferred embodiments the bacterial cell canbe a Porphyromonas (Bacteroides) gingivalis; Bacteroides speciesincluding B. gingivalis (now known as Porphyromonas gingivalis),Eikenella corrodens, Fusobacterium nucleatum, Wolinella recta,Eubacterium species, Prevotella (Bacteroides) intermedia, Bacteroidesforsythus, Capnocytophaga species, Actinobacillus actinomycetamcomitans,and Streptococcus mutans.

In preferred embodiments where the bacterial cell is a Treponema cell,the disorder is trenchmouth, yaws, or pinta. In other embodiments thedisorder is impetigo or cystic acne.

In preferred embodiments where the unwanted organism is a fungal cell,the cell can be a Candida or an Aspergillus cell. In preferredembodiments, the organism is Pneumocystis carinii.

In preferred embodiments where the unwanted organism is a protozoancell, the cell is an Entamoeba, a Toxoplasma, a Giardia, a Leishmania, aCrytosporidium, or a Schistosoma.

In preferred embodiments where the unwanted organism is a virus, thevirus is an HIV, an HTLV, a hepatitis virus, an influenza virus, arhinovirus, a papilloma virus, a measles virus, a Herpes virus, arotavirus, a parvovirus, a psittacosis virus, or an Ebola virus.

In preferred embodiments where the target organism is an arthropod, thearthropod is a parasitic mite.

In preferred embodiments where the target organism is a helminth, thehelminth is a nematode or a trematode. In preferred embodiments wherethe helminth is a nematode, the nematode is found in a subject withfilariasis.

In preferred embodiments the target organism is an oral bacterialspecies, e.g., Porphyromonas (Bacteroides) gingivalis.

In preferred embodiments, the conjugate does not include, e.g., it isnot coupled, e.g., covalently or non-covalently coupled to: a PM; anantibody; an enzyme; a hormone; a receptor on a cell surface; or theligand for a receptor on a cell surface.

In another aspect, the invention features, a method of treating asubject, for a disorder of the oral cavity characterized by the presenceof an unwanted organism. The method includes:

administering to the subject, a conjugate which includes aphotosensitizer coupled to a NPM targeting moiety, e.g., a conjugatedescribed herein;

irradiating the subject with energy of a wavelength appropriate toproduce a cytotaxic effect by the photosensitizer;

thereby treating the subject, for the disorder characterized by thepresence of an unwanted organism.

In preferred embodiments the method includes topically administering theconjugate to an area of the subject which is infected with the unwantedorganisms. The conjugate can be topically administered e.g., generallyto the surfaces of the oral cavity, to the gums, to the periodontaltissue, to the periodontal pocket, to areas characterized byinflammation, to lesions, to fissures or imperfections in a tooth orgum, to dental carries, to cuts or incisions, e.g., those made in thecourse of dental or other medical care, or to wounds. In otherembodiments the method includes systemic administration, e.g., byingestion or injection. In other embodiments the method includessubcutaneous delivery, e.g., subcutaneous injection. In otherembodiments the method includes local injection at or near the site ofinfection with the unwanted organism.

In preferred embodiments the radiation: is laser irradiation; or isdelivered with a fiber optic devise.

In preferred embodiments the subject is suffering from: a disorder ofthe oral cavity which is characterized by the presence of an unwantedorganism, e.g., a microbial organism, e.g., an unwanted bacterium,fungus, virus, or protozoan. The disorder can be one in which any of theteeth, gums, e.g., the periodontal tissue, tongue, tonsils, uvula,lining of the oral cavity, or parotid glands, are infected by theorganism or otherwise affected by the disorder.

In preferred embodiments the disorder is an infectious oral disease.

In preferred embodiments the subject is suffering from: a periodontaldisease, e.g., periodontitis or periodontosis; receding gums; acuteulcerative gingivitis; chronic gingivitis; periodontal abscess; earlyonset (juvenile) periodontitis; gingivitis of pregnancy; pericoronitis;infective stomatitis; cancrum oris; suppurative paratitis; acute orchronic osteomyelitis of the mandibles or maxilla; pulpitis orperiapical infections.

In preferred embodiments the subject is suffering from an oral fungalinfection, e.g., an actinomycosis, histoplasmosis, phycomycosis,aspergillosis, cryptococcosis, sporotrichosis, blastomycosis, orparacoccidioidomycosis infection.

In other preferred embodiments, the subject is suffering from an oralyeast infection, e.g., including a Candida infection of the oral cavity,e.g., candidosis (candidiasis), thrush, chronic candidosis and candidal(candididal) leukoplakia, or from a viral infection including primaryherpetic stomatitis, or herpes labialis.

In preferred embodiments the subject, in addition to suffering from adisorder of the oral cavity, is suffering from an immune disorder, e.g.,an acquired or inherited immune disorder. In particularly preferredembodiments the subject is suffering from AIDS or is HIV positive.

In preferred embodiments the unwanted organism is: an oral bacterialspecies, e.g., Porphyromonas (Bacteroides) gingivalis; Bacteroidesspecies including B. gingivalis (now known as Porphyromonas gingivalis),Eikenella corrodens, Fusobacterium nucleatum, Wolinella recta,Eubacterium species, Prevotella (Bacteroides) intermedia, Bacteroidesforsythus, Capnocytophaga species, Actinobacillus actinomycetamcomitans,and Streptococcus mutans.

In preferred embodiments, the targeting moiety of the conjugate includesa salivary polypeptide, or an active fragment or analog thereof.Examples of salivary polypeptides are the histatins, e.g., histatin-1through -8, or more preferably, histatin-1, -3, or -5. In preferredembodiments the targeting moiety includes histatin-5 residues 13-24, orcorresponding residues from other histatins. In preferred embodimentsthe targeting moiety includes a histatin molecule which has beenengineered to include an internal duplication.

In particularly preferred embodiments the targeting moiety includes apolylysine molecule.

In preferred embodiments the targeting moiety includes a polypeptide,e.g., a polyamino acid, which has been chemically modified to alter itscharge, e.g., the charge of side chains of one or more amino acidresidues of the polyamino acid. For example, one or more, orapproximately 10, 25, 50, 75, 90 or 100% of the charged side chains canbe modified. By modified is meant that a negative side chain, e.g., aglutamic acid, or an aspartic acid, side chain is made positive orneutral in charge, a positively charged side chain, e.g., the side chainof lysine, arginine, or ornithine is made negative or neutral in charge.By way of example, one or more of the side chains of polylysine can bemade neutral or negative in charge.

In preferred embodiments, the conjugate does not include, e.g., it isnot coupled, e.g., covalently or non-covalently coupled to: a PM; anantibody; an enzyme; a hormone; a receptor on a cell surface; or theligand for a receptor on a cell surface.

In preferred embodiments: the photosensitizer produces singlet oxygenupon absorption of electromagnetic irradiation at the proper energylevel and wavelength; the photosensitizer includes a porphyrin orporphyrin derivative; the photosensitizer includes chlorin e6 or achlorin derivative.

In another aspect, the invention features a method of treating a subjectfor a periodontal disorder characterized by the presence of an unwantedorganism. The method includes:

administering to the subject, a conjugate which includes aphotosensitizer coupled to a NPM targeting moiety, e.g., a conjugatedescribed herein;

irradiating periodontal tissue of the subject with energy of awavelength appropriate to produce a cytotaxic effect by thephotosensitizer;

thereby treating the subject, for the periodontal disorder.

In preferred embodiments the method includes topically administering theconjugate to an area of the subject which is infected with the unwantedorganisms. The conjugate can be topically administered to the gums, tothe periodontal tissue, to the periodontal pocket, to areascharacterized by inflammation, or lesions. In other embodiments themethod includes systemic administration, e.g., by ingestion orinjection. In other embodiments the method includes subcutaneousdelivery, e.g., subcutaneous injection. In other embodiments the methodincludes local injection at or near the site of infection with theunwanted organism.

In preferred embodiments the subject is suffering from: periodontitis orperiodontosis; receding gums; acute ulcerative gingivitis; chronicgingivitis; periodontal abscess; early onset (Juvenile) periodontitis;gingivitis of pregnancy.

In preferred embodiments the subject, in addition to suffering from aperiodontal disorder, is suffering from an immune disorder, e.g., anacquired or inherited immune disorder. In particularly preferredembodiments the subject is suffering from AIDS or is HIV positive.

In preferred embodiments the unwanted organism is: an oral bacterialspecies, e.g., Porphyromonas (Bacteroides) gingivalis; Bacteroidesspecies including B. gingivalis (now known as Porphyromonas gingivalis),Eikenella corrodens, Fusobacterium nucleatum, Wolinella recta,Eubacterium species, Prevotella (Bacteroides) intermedia, Bacteroidesforsythus, Capnocytophaga species, Actinobacillus actinomycetamcomitans,and Streptococcus mutans.

In preferred embodiments, the targeting moiety of the conjugate includesa salivary polypeptide, or an active fragment or analog thereof.Examples of salivary polypeptides are the histatins, e.g., histatin-1through -8, or more preferably, histatin-1, -3, or -5. In preferredembodiments the targeting moiety includes histatin-5 residues 13-24, orcorresponding residues from other histatins. In preferred embodimentsthe targeting moiety includes a histatin molecule which has beenengineered to include an internal duplication.

In particularly preferred embodiments the targeting moiety includes apolylysine molecule.

In preferred embodiments the targeting moiety includes a polypeptide,e.g., a polyamino acid, which has been chemically modified to alter itscharge, e.g., the charge of side chains of one or more amino acidresidues of the polyamino acid. For example, one or more, orapproximately 10, 25, 50, 75, 90 or 100% of the charged side chains canbe modified. By modified is meant that a negative side chain, e.g., aglutamic acid, or an aspartic acid, side chain is made positive orneutral in charge, a positively charged side chain, e.g., the side chainof lysine, arginine, or ornithine is made negative or neutral in charge.By way of example, one or more of the side chains of polylysine can bemade neutral or negative in charge.

In preferred embodiments, the conjugate does not include, e.g., it isnot coupled, e.g., covalently or non-covalently coupled to: a PM; anantibody; an enzyme; a hormone; a receptor on a cell surface; or theligand for a receptor on a cell surface.

In preferred embodiments: the photosensitizer produces singlet oxygenupon absorption of electromagnetic irradiation at the proper energylevel and wavelength; the photosensitizer includes a porphyrin orporphyrin derivative; the photosensitizer includes chlorin e6 or achlorin derivative.

In another aspect, the invention features, a method of treating asubject having an acquired immune disorder having an acquired immunedisorder, for a disorder of the oral cavity characterized by thepresence of an unwanted organism. In preferred embodiments the unwantedorganism is other than an organism which is causative of the acquiredimmune disorder. The acquired immune disorder can be, e.g., AIDS, or anHIV infection. The method includes:

administering to the subject, a conjugate which includes aphotosensitizer coupled to a NPM targeting moiety, e.g., a conjugatedescribed herein;

irradiating the subject with energy of a wavelength appropriate toproduce a cytotaxic effect by the photosensitizer;

thereby treating the subject, for the disorder characterized by thepresence of an unwanted organism.

In preferred embodiments the method includes topically administering theconjugate to an area of the subject which is infected with the unwantedorganisms. The conjugate can be topically administered e.g., generallyto the surfaces of the oral cavity, to the gums, to the periodontaltissue, to the periodontal pocket, to areas characterized byinflammation, to lesions, to fissures or imperfections in a tooth orgum, to dental carries, to cuts or incisions, e.g., those made in thecourse of dental or other medical care, or to wounds. In otherembodiments the method includes systemic administration, e.g., byingestion or injection. In other embodiments the method includessubcutaneous delivery, e.g., subcutaneous injection. In otherembodiments the method includes local injection at or near the site ofinfection with the unwanted organism.

In preferred embodiments the radiation: is laser irradiation; or isdelivered with a fiber optic devise.

In preferred embodiments the unwanted organism is, e.g., a microbialorganism, e.g., an unwanted bacterium, fungus, virus, or protozoan. Thedisorder can be one in which any of the teeth, gums, e.g., theperiodontal tissue, tongue, tonsils, uvula, lining of the oral cavity,parotid glands, are infected by the organism or otherwise affected bythe disorder.

In preferred embodiments the disorder is an infectious oral disease.

In preferred embodiments the subject is suffering from: a periodontaldisease, e.g., periodontitis or periodontosis; receding gums; acuteulcerative gingivitis; chronic gingivitis; periodontal abscess; earlyonset juvenile) periodontitis; gingivitis of pregnancy; periocoronities;infective stomatitis; cancrum oris; suppurative paratitis; acute orchronic osteomyelitis of the mandibles or maxilla; pulpitis orperioapical infections.

In preferred embodiments the subject is suffering from an oral fungalinfection, e.g., an actinomycosis, histoplasmosis, phycomycosis,aspergillosis, cryptococcosis, sporotrichosis, blastombycosis, orparacoccidioidomycosis infection. In other preferred embodiments, thesubject is suffering from an oral yeast infection, e.g., including aCandida infection of the oral cavity, e.g., candidosis (candidiasis),thrush, chronic candidosis and candidal (candididal) leukoplakia, orfrom a viral infection including primary herpetic stomatitis, or herpeslabialis.

In preferred embodiments the unwanted organism is: an oral bacterialspecies, e.g., Porphyromonas (Bacteroides) gingivalis; Bacteroidesspecies including B. gingivalis (now known as Porphyromonas gingivalis),Eikenella corrodens, Fusobacterium nucleatum, Wolinella recta,Eubacterium species, Prevotella (Bacteroides) intermedia, Bacteroidesforsythus, Capnocytophaga species, Actinobacillus actinomycetamcomitans,and Streptococcus mutans.

In preferred embodiments, the targeting moiety of the conjugate includesa salivary polypeptide, or an active fragment or analog thereof.Examples of salivary polypeptides are the histatins, e.g., histatin-1through -8, or more preferably, histatin-1, -3, or -5. In preferredembodiments the targeting moiety includes histatin-5 residues 13-24, orcorresponding residues from other histatins. In preferred embodimentsthe targeting moiety includes a histatin molecule which has beenengineered to include an internal duplication.

In particularly preferred embodiments the targeting moiety includes apolylysine molecule.

In preferred embodiments the targeting moiety includes a polypeptide,e.g., a polyamino acid, which has been chemically modified to alter itscharge, e.g., the charge of side chains of one or more amino acidresidues of the polyamino acid. For example, one or more, orapproximately 10, 25, 50, 75, 90 or 100% of the charged side chains canbe modified. By modified is meant that a negative side chain, e.g., aglutamic acid, or an aspartic acid, side chain is made positive orneutral in charge, a positively charged side chain, e.g., the side chainof lysine, arginine, or ornithine is made negative or neutral in charge.By way of example, one or more of the side chains of polylysine can bemade neutral or negative in charge.

In preferred embodiments, the conjugate does not include, e.g., it isnot coupled, e.g., covalently or non-covalently coupled to: a PM; anantibody; an enzyme; a hormone; a receptor on a cell surface; or theligand for a receptor on a cell surface.

In preferred embodiments: the photosensitizer produces singlet oxygenupon absorption of electromagnetic irradiation at the proper energylevel and wavelength; the photosensitizer includes a porphyrin orporphyrin derivative; the photosensitizer includes chlorin e6 or achlorin derivative.

In another embodiment, the invention includes a method for makingconjugate molecules, the method comprising:

supplying a backbone, e.g., a polypeptide backbone;

coupling, e.g., covalently coupling, a photosensitizer to the backbone;

coupling, e.g., covalently coupling, a targeting moiety, e.g., atargeting moiety described herein, to the backbone.

In preferred embodiments, the coupling reactions involve an activatedester moiety of a photosensitizer. In more preferred embodiments, anamino group on the backbone reacts as a nucleophile, displacing theleaving group from the photosensitizer active ester. In preferredembodiments, the targeting moiety is coupled to the backbone with acoupling agent.

In preferred embodiments, the conjugate does not include, e.g., it isnot coupled, e.g., covalently or non-covalently coupled to: a PM; anantibody; an enzyme; a hormone; a receptor on a cell surface; or theligand for a receptor on a cell surface.

In another aspect, the invention features, a kit for elimination of anunwanted organism. The kit includes a photosensitizer coupled to atargeting moiety and instructions for use.

In preferred embodiments, the conjugate does not include, e.g., it isnot coupled, e.g., covalently or non-covalently coupled to: a PM; anantibody; an enzyme; a hormone; a receptor on a cell surface; or theligand for a receptor on a cell surface.

Photodynamic therapy involves the use of a light activatable compound,or photosensitizer, together with light of the correct wavelength, toproduce a cytotoxic effect. In order to increase the specificity of thephotosensitizer for its target, the photosensitizer may be bound to atargeting moiety. Methods and conjugates of the invention features theuse of NPM-targeted photosensitizers. NPM's can deliver photosensitizerto a target in an efficient and cost effective manner. Compositions ofthe invention are advantageous in that (i) they do not need to beinternalized to kill bacteria, since illumination generates toxic oxygenspecies which can diffuse through the bacterial cell wall, (ii) thegeneration of toxic oxygen species can have a local effect instimulating the host immune response which can assist in eradicatingbacteria and in promoting healing of the wound, (iii) they produce acytotoxic response only in the area subject to illumination.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those herein can be used in the practice ortesting of the present invention, the preferred methods and materialsare described below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a diagram of the first of the reactions for synthesis ofchlorin e6 polylysine conjugates of different charge, with arrow 1showing the reaction of chlorin e6, molecule I, withN-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide anddimethyl sulfoxide to form chlorin e6-NHS ester, molecule II.

FIG. 1B is a diagram showing reaction 2 of molecule II withpoly-L-lysine, molecule IV, to form poly-L-lysine chlorin e6, moleculeIII.

FIG. 1C is a diagram showing the chemical structure of histatin-5chlorin e6 conjugate, with four chlorin e6 moieties each coupled to anε-amino group of each lysine residue of histatin-5.

FIG. 1D is a diagram showing the reaction of molecule III with aceticanhydride or succinic anhydride in DMSO to form two species of moleculeV, which for chlorin e6 polylysine acetic acid amide neutral conjugatethe R group indicates —COCH₃, and for chlorin e6 polylysine succinicacid amide the R group indicates —COCH₂CH₂COOH; for molecule III that isnot further acylated, the R group in species V is —H.

FIG. 2A is a diagram of the first reaction sequences for synthesis ofcationic, neutral and anionic chlorin e6 polylysine conjugates with ahistatin-5 16 amino acid residue fragment, by synthesis of moleculescontaining the sulfhydryl reactive functional group2-pyridyl-dithio-3-propionyl, with reaction 4 showing molecule V,polylysine-chlorin e6, reacting with N-succinimidyl-3-[2-pyridyldithiol]propionate (SPDP) in DMSO to produce polylysine-chlorin e6-SPDP,molecule VI, and with further reaction 5 of molecule VI with aceticanhydride or succinic anhydride in DMSO to produce the acetyl andsuccinyl derivatives, where R in molecule VII indicates —COCH₃ and—COCH₂CH₂COOH, respectively.

FIG. 2B is a diagram that shows further reactions of molecules of VIIand of a 16 amino acid residue fragment of human histatin-5 tosynthesize the conjugate, with reaction 6 showing reaction of thehistatin-5 fragment with SATA to produce SATA-histatin-5 fragment,molecule IX, which is then deprotected by hydroxylamine in reaction 7 toproduced deprotected SATA coupled to histatin-5 fragment, molecule X.

FIG. 2C is a diagram showing reaction 8 of molecule X which is herecoupled to each of the polylysine-chlorin e6 molecules and acetyl andsuccinyl derivatives of molecule VII, to produce the molecular speciesin XI, where R indicates —H for the positively charged unacylatedconjugate, R indicates —COCH₃ for the neutral acetic acid amide, and Rindicates —COCH₂CH₂COOH for the negatively charged succinic acid amide.

FIG. 3 is a graph of uptake of three polylysine chlorin e6 conjugates,in 10⁻⁹ mol of chlorin e6 per mg of cell protein by cells ofPorphyromonas gingivalis, with uptake of the cationic conjugate shown bythe solid line connecting circles, uptake of the anionic succinylatedconjugate shown by the dashed line connecting squares, and uptake of theneutral acetylated conjugate shown by the dotted line connecting solidcircles, as a function of chlorin concentration in μM.

FIG. 4 is a graph of uptake of the three chlorin e6 conjugates, in 10⁻¹¹mol of chlorin e6 per mg of cell protein by cells of HCPC-1 hamstercheek pouch squamous cell carcinoma cells as a function of chlorinconcentration in μM, with symbols representing each conjugate asdescribed in the legend to FIG. 3.

FIG. 5 is a graph of survival of P. gingivalis cells followingirradiation by light of wavelength 630-710 nm, the cells previouslyhaving taken up chlorin e6 conjugates according to the symbols indicatedin the legend to FIG. 3, with an additional cell sample having taken upPhotofrin II (shown by a dashed line connecting the x symbols), andanother cell sample having taken up benzoporphyrin derivative (shown bya dashed heavy line connecting solid triangles), as a function offluence of light.

FIG. 6 is a graph of survival of HCPC-1 hamster squamous cell carcinomacells following irradiation by light of wavelength 630-710 nm, cellspreviously having taken up chlorin e6 conjugates as indicated by thesymbols in FIGS. 3 and 6, as a function of fluence of light.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, a photosensitizer refers to a substance which, uponirradiation with electromagnetic energy of the appropriate wavelength,usually light of the appropriate wavelength, produces a cytotoxiceffect.

As used herein, the terms peptide, polypeptide, and protein are, unlessspecified otherwise, used interchangeably. Peptides, polypeptides, andproteins used in methods and compositions described herein can berecombinant, purified from natural sources, or chemically synthesized.For example, reference to the use of a bacterial protein or a proteinfrom bacteria, includes the use of recombinantly produced molecules,molecules purified from natural sources, or chemically synthesizedmolecules.

As used herein, a non-pair-member (NPM) is a molecule, e.g., apolypeptide, which binds to a target site. A target site can be aprotein, nucleic acid, lipid, polysaccharide, or a structure which is acombination thereof. Antibodies, receptors, hormones, growth factors,neurotransmitters and enzymes are not NPM polypeptides and are referredto herein as pair-members (PM's). NPM's do not exhibit a complementaryrelationship between the NPM and the binding site. By complementaryrelationship is meant that the two entities which bind, have acomplementary combination of shape, contour, and charge which is basedon the ability of “opposing” functional groups located on one entitybeing capable of forming non-covalent bonds with “opposing” functionalgroups located on the other entity, thereby complexing the two entitieswith a plurality of non-covalent interactions. In other words, the NPMwill not contain a combination of shape, contours, and charge patternsthat are complementary to those of the target site. Generally, a NPMwill have lower affinity for its target than do PM's, e.g., the PM'sdescribed above. Typical affinities of NPM's for their targets are inthe Km range values of mM to μM. Release of the binding of PM's, forexample, an enzyme from a substrate, in vivo, often requires the bindingof an additional protein, or the breaking or forming of a covalent bond.In vitro, PM pairs are often manipulated to separate into componententities by extremes of heat or pH.

Methods of the invention can use a targeting moiety which has a PMfunction in addition to the binding characteristic to be used for thepurposes of the instant invention. For example, LDL molecules bind asPM-type ligands to apo B/E classical LDL receptors, and oxidized orotherwise altered LDL molecules bind as PM-type ligands to macrophagescavenge receptors. However, LDL molecules have, in addition, anNPM-type affinity for Gram negative surface components, which is not aPM function, as defined herein. PM interactions are generallycharacterized by high specificity, so that only one or a few cognatemolecules are recognized, and high affinity, with Km values generally inthe range of μM to pM.

As used herein, target organism, means an organism which causes oraggravates a disorder.

The term “subject,” as used herein, refers to a living animal or humancarrying an unwanted organism, that is, an organism that is a target forphotodynamic therapy. The subject may be immune deficient. The subjectcan be a mammal, including humans and non-human mammals such as dogs,cats, pigs, cows, sheep, goats, horses, rats, and mice. The subject mayformerly have been treated by chemotherapy or antibiotic therapy.

As used herein, “naturally occurring” refers to a molecule which existsin nature. A non-naturally occurring molecule is one having a structurewhich does not occur without human intervention.

As used herein, the term small anti-microbial peptide (SAMP) refers to apeptide of less than 60 amino acid residues in length. Histatins,defensins, cecropins, magainins, Gram positive bacteriocins, and peptideantibiotics which meet this limitation are SAMP's. Many SAMP's are inthe range of 20-40 amino acid residues in length. SAMP's are naturallyoccurring peptides, and are made by a wide variety of organisms. SAMP'sare NPM's. Many SAMP's have a broad spectrum of antimicrobial activity,and, e.g., can kill more than one species, and in some cases can killdistantly related species, e.g. Gram negative and Gram positivebacterial species.

As used herein, an active fragment or analog of a polypeptide is onewhich retains at least 20% of the antimicrobial activity or targetorganism affinity of the polypeptide. Analogs of a polypeptide share atleast 50% and more preferably 60, 70, 80 or 90% sequence identity withthe polypeptide.

A salivary polypeptide, as used herein, refers to a polypeptide producedby a subject and found in the subject's saliva. Most salivarypolypeptides are produced by the parotid gland.

As used herein, a peptide antibiotic is a linear or cyclic oligopeptide,or an active fragment, or analog thereof, which possesses antibioticactivity against bacterial or fungal species, and which is synthesizedenzymatically on a multi-protein complex to which it is attached by athioether bond. A peptide antibiotic may include non-ribosomal aminoacids such as D amino acids, and may include non-amino acid residuessuch as esters of lactic acid or valeric acid.

Photosensitizers

A photosensitizer is a substance which, upon irradiation withelectromagnetic energy of the appropriate wavelength, usually light ofthe appropriate wavelength, produces a cytotoxic effect.

Many photosensitizers produce singlet oxygen. Upon electromagneticirradiation at the proper energy level and wavelength, such aphotosensitizer molecule is converted to an energized form. Theenergized form can react with atmospheric O₂, such that upon decay ofthe photosensitizer to the unenergized state, singlet oxygen isproduced. Singlet oxygen is highly reactive, and is toxic to a proximaltarget organism.

The life-time of its triplet energized state should be of sufficientduration (e.g., several microseconds) to permit interaction withneighboring molecules to produce cytotoxic species.

A photosensitizer composition should efficiently absorb electromagneticenergy of the appropriate wavelength with high quantum yield toefficiently generate the energized form of the photosensitizer. Toxicityto the target organism should increase substantially, preferably10-fold, 100-fold, or even more preferably 1,000-fold, upon irradiation.A photosensitizer should exhibit low background toxicity, i.e., lowtoxicity in the absence of irradiation with energy of the appropriatewavelength.

Other preferred properties of a photosensitizer include high solubilityand stability in appropriate solvents. For example, a photosensitizershould be soluble under conditions used to couple it to the targetingmoiety or backbone. Desired solubility properties will differ with theconditions chosen for the reaction but solubility in DMSO, water,ethanol, or a mixture of water and DMSO or in ethanol, such as DMSO:H₂O, or in ethanol:water 5%, 10% or 15% can be useful. Solubility ispreferably 50 μg/ml, 100 μg/ml, 500 μg/ml, 1 mg/ml or 10 mg/ml in anaqueous solvent or ethanol:water solvent.

When conjugated to a targeting moiety, the resultingphotosensitizer:targeting moiety conjugate should be soluble underphysiological conditions, in aqueous solvents containing appropriatecarriers and excipients, or other delivery systems such as in liposomes.The molecules of the invention may be delivered as freephotosensitizer:targeting moiety compositions in solution, and may bedelivered also in various formulations including, but not limited to,liposome, peptide/polymer-bound, or detergent-containing formulations.

The compositions of the invention should be stable during the course ofat least a single round of treatment by continuous or pulsedirradiation, during which each molecule of the composition wouldpreferably be repeatedly excited to the energized state, undergoingmultiple rounds of generation of singlet oxygen. Preferable stability ofa photosensitizer conjugate molecule is survival of 10%, 50%, 90%, 95%,or 99% of molecules in active form for 1 hour, for 30 min, 15 min or forat least 1 min at 37° C., under physiological conditions.

Photosensitizers include, but are not limited to, hematoporphyrins, suchas hematoporphyrin HCl and hematoporphyrin esters (Dobson, J. and M.Wilson, Archs. Oral Biol. 37:883-887); dihematophorphyrin ester (Wilson,M. et al., 1993, Oral Microbiol. Immunol. 8:182-187); hematoporphyrin IX(Russell et al., 1991, Can J. App. Spectros. 36:103-107, available fromPorphyrin Products, Logan, Utah) and its derivatives; 3,1-mesotetrakis(o-propionamidophenyl)porphryrin; hydroporphyrins such aschlorin, herein, and bacteriochlorin of thetetra(hydroxyphenyl)porphyrin series, and synthetic diporphyrins anddichlorins; o-substituted tetraphenyl porphyrins (picket fenceporphyrins); chlorin e6 monoethylendiamine monamide (CMA Goff, B. A. etal., 1994, 70:474-480, available from Porphyrin Products, Logan, Utah);mono-1-aspartyl derivative of chlorin e6, and mono- and di-aspartylderivatives of chlorin e6; the hematoporphyrin mixture Photofrin II(Quardra Logic Technologies, Inc., Vancouver, BC, Canada);benzophorphyrin derivatives (BPD), including benzoporphyrin monoacidRing A (BPD-MA), tetracyanoethylene adducts, dimethyl acetylenedicarboxylate adducts, Diels-Adler adducts, and monoacid ring “a”derivatives; a naphthalocyanine (Biolo, R., 1994, Photochem. andPhotobiol. 5959:362-365); a Zn(II)-phthalocyanine (Shopora, M. et al.,1995, Lasers in Medical Science 10:43-46); toluidine blue O (Wilson, M.et al., 1993, Lasers in Medical Sci. 8:69-73); aluminum sulfonated anddisulfonated phthalocyanine ibid.; and phthalocyanines without metalsubstituents, and with varying other substituents; a tetrasulfatedderivative; sulfonated aluminum naphthalocyanines; methylene blue(ibid.); nile blue; crystal violet; azure β chloride; and rose bengal(Wilson, M., 1994, Intl. Dent. J. 44:187-189). Numerous photosensitizerentities are disclosed in Wilson, M. et al., 1992, Curr. Micro.25:77-81, and in Okamoto, H. et al., 1992, Lasers in Surg. Med.12:450-485.

Other potential photosensitizer compositions include but are not limitedto, pheophorbides such as pyropheophorbide compounds, anthracenediones;anthrapyrazoles; aminoanthraquinone; phenoxazine dyes; phenothiazinederivatives; chalcogenapyrylium dyes including cationic selena- andtellura-pyrylium derivatives; verdins; purpurins including tin and zincderivatives of octaethylpurpurin and etiopurpurin;benzonaphthoporphyrazines; cationic imminium salts; and tetracyclines.

The suitability of a photosensitizer for use in a conjugate can bedetermined by methods described herein or by methods known to thoseskilled in the art.

The efficiency with which a photosensitizer oxidizes a target moleculeis a measure of the usefulness. The efficiency of the oxidation of atarget molecule by a photosensitizer can be determined in vitro.Examples of substrates include 4-nitroso-N,N-dimethylaniline (RNO;Hasan, T. et al., 1987, Proc. AACR 28:395 Abstr. 1,568), and tryptophanor histidine (Lambert, C. R. et al., 1986, Photochem. Photobiol.44:595-601). In these assays, ability of a candidate photosensitizer to“bleach” the substrate can be monitored spectroscopically. The advantageof a chemical assay is that a large number of putative photosensitizercompositions can be simultaneously screened. Parameters which can bevaried include photosensitizer concentration, substrate concentration,optimal intensity of irradiation, and optimal wavelength of irradiation.High through-put technologies including plastic multi-well dishes,automated multi-pipetters, and on-line spectrophotometric plate readerscan be used. Undesirable candidates, e.g., compositions having highbackgrounds under unirradiated conditions, inefficient energy capture orreactive potential, can be identified and eliminated.

In vivo assays with cells of one or more model target organisms can beused to evaluate a photosensitizer for cytotoxicity of its backgroundand activated forms. The efficiency of killing of the organism in thepresence of the irradiated and unirradiated photosensitizer can bemeasured and compared to survival of the untreated control cell sample.This assay can be automated. The use of counts of colony forming units(CFU) or cell growth may require incubation of the samples that havebeen applied to a nutrient medium, with a concomitant lag of theappropriate growth period to allow for colony formation.

Survival of cells of the model target organism can alternatively bemonitored by assay of a biochemical process, for example, assay of DNAsynthesis. In this approach the effectiveness of a photosensitizercandidate can be measured by its effect on samples of cells of the modelorganism, which are also exposed to a labeled DNA precursor such astritiated thymidine. Cells are then collected, washed to removeunincorporated precursor, and monitored for uptake of the precursor andincorporation into acid-insoluble precipitate, which is a measure ofquantity of DNA synthesis. In this assay, which can also be automated asdescribed above, quantitative evaluation of the effects of presence ofirradiated photosensitizer compositions can be readily evaluated andquantitated. In control unirradiated cells and in untreated cells, DNAsynthesis increases logarithmically as a function of cell growth. Apositive result indicating presence of a putative successful novelphotosensitizer, is turn-off of DNA synthesis in cells that have beenirradiated in the presence of that photosensitizer.

Suitable model target organisms are: Escherichia coli, Pseudomonasaeruginosa, Staphylococcus aureus and Streptococcus mutans. A suitablepositive control for photosensitizer activity is toluidine blue O.

If large numbers of candidates are to be screened it may be desirable touse a two-stage screen, wherein the first stage is an in vitro screenand wherein the second stage uses cells.

Irradiation

Irradiation of the appropriate wavelength for a given compound may beadministered by a variety of methods. These methods include but are notlimited to the administration of laser, nonlaser, or broad band light.Irradiation can be produced by extracorporeal or intraarticulargeneration of light of the appropriate wavelength. Light used in theinvention may be administered using any device capable of delivering therequisite power of light including, but not limited to, fiber opticinstruments, arthroscopic instruments, or instruments which providetransillumination. Delivery of light to the oral cavity can beaccomplished with flexible fiber optics which are inserted into theperiodontal pocket, or by transgingival illumination (average thicknessof gingiva is 5-7 mm). The source of the light needed to inactivate thebacteria can be an inexpensive diode laser or a non-coherent lightsource.

Coupling Technologies

The term “coupling agent” as used herein, refers to a reagent capable ofcoupling a photosensitizer to a targeting moiety, or a photosensitizeror a targeting moiety to a “backbone” or “bridge” moiety. Any bond whichis capable of linking the components such that they are stable underphysiological conditions for the time needed for administration andtreatment is suitable, but covalent linkages are preferred. The linkbetween two components may be direct, e.g., where a photosensitizer islinked directly to a targeting moiety, or indirect, e.g., where aphotosensitizer is linked to an intermediate, e.g., linked to abackbone, and that intermediate being linked to the targeting moiety. Acoupling agent should function under conditions of temperature, pH,salt, solvent system, and other reactants that substantially retain thechemical stability of the photosensitizer, the backbone (if present),and the targeting moiety.

A coupling agent can link components without the addition to the linkedcomponents of elements of the coupling agent. Other coupling agentsresult in the addition of elements of the coupling agent to the linkedcomponents. For example, coupling agents can be cross-linking agentsthat are homo- or hetero-bifunctional, and wherein one or more atomiccomponents of the agent can be retained in the composition. A couplingagent that is not a cross-linking agent can be removed entirely duringthe coupling reaction, so that the molecular product can be composedentirely of the photosensitizer, the targeting moiety, and a backbonemoiety (if present).

Many coupling agents react with an amine and a carboxylate, to form anamide, or an alcohol and a carboxylate to form an ester. Coupling agentsare known in the art, see, e.g., M. Bodansky, “Principles of PeptideSynthesis”, 2nd ed., referenced herein, and T. Greene and P. Wuts,“Protective Groups in Organic Synthesis,” 2nd Ed, 1991, John Wiley, NY.Coupling agents should link component moieties stably, but such thatthere is only minimal or no denaturation or deactivation of thephotosensitizer or the targeting moiety.

The photosensitizer conjugates of the invention can be prepared bycoupling the photosensitizer to targeting moieties using methodsdescribed in the following Examples, or by methods known in the art. Avariety of coupling agents, including cross-linking agents, can be usedfor covalent conjugation. Examples of cross-linking agents includeN,N′-dicyclohexylcarbodiimide (DCC; Pierce),N-succinimidyl-S-acetyl-thioacetate (SATA),N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),ortho-phenylenedimaleimide (o-PDM), and sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC). See, e.g.,Karpovsky et al. J. Exp. Med. 160:1686, 1984; and Liu, MA et al., Proc.Natl. Acad. Sci. USA 82:8648, 1985. Other methods include thosedescribed by Paulus, Behring Ins. Mitt., No. 78, 118-132, 1985; Brennanet al. Science 229:81-83, 1985, and Glennie et al., J. Immunol., 139:2367-2375, 1987. A large number of coupling agents for peptides andproteins, along with buffers, solvents, and methods of use, aredescribed in the Pierce Chemical Co. catalog, pages T-155-T-200, 1994(3747 N. Meridian Rd., Rockford Ill., 61105, U.S.A.; Pierce Europe B.V.,P.O. Box 1512, 3260 BA Oud Beijerland, The Netherlands), which catalogis hereby incorporated by reference.

DCC is a useful coupling agent (Pierce #20320; Rockland, Ill.). Itpromotes coupling of the alcohol NHS to chlorin e6 in DMSO (Pierce#20684), forming an activated ester which can be cross-linked topolylysine. DCC (N,N′-dicyclohexylcarbodiimide) is a carboxy-reactivecross-linker commonly used as a coupling agent in peptide synthesis, andhas a molecular weight of 206.32. Another useful cross-linking agent isSPDP (Pierce #21557), a heterobifunctional cross-linker for use withprimary amines and sulfhydryl groups. SPDP has a molecular weight of312.4, a spacer arm length of 6.8 angstroms, is reactive to NHS-estersand pyridyldithio groups, and produces cleavable cross-linking suchthat, upon further reaction, the agent is eliminated so thephotosensitizer can be linked directly to a backbone or targetingmoiety. Other useful conjugating agents are SATA (Pierce #26102) forintroduction of blocked SH groups for two-step cross-linking, which isdeblocked with hydroxylamine-HCl (Pierce #26103), and sulfo-SMCC (Pierce#22322), reactive towards amines and sulfhydryls. Other cross-linkingand coupling agents are also available from Pierce Chemical Co.(Rockford, Ill.); Additional compounds and processes, particularly thoseinvolving a Schiff base as an intermediate, for conjugation of proteinsto other proteins or to other compositions, for example to reportergroups or to chelators for metal ion labeling of a protein, aredisclosed in EPO 243,929 A2 (published Nov. 4, 1987).

Photosensitizers which contain carboxyl groups can be joined to lysineε-amino groups in the target polypeptides either by preformed reactiveesters (such as N-hydroxy succinimide ester) or esters conjugated insitu by a carbodiimide-mediated reaction. The same applies tophotosensitizers which contain sulfonic acid groups, which can betransformed to sulfonyl chlorides which react with amino groups.Photosensitizers which have carboxyl groups can be joined to aminogroups on the polypeptide by an in situ carbodiimide method.Photosensitizers can also be attached to hydroxyl groups, of serine orthreonine residues or to sulfhydryl groups of cysteine residues.

Methods of joining components of a conjugate, e.g., coupling polyaminoacid chains bearing photosensitizers to antibacterial polypeptides, canuse heterobifunctional cross linking reagents. These agents bind afunctional group in one chain and to a different functional group in thesecond chain. These functional groups typically are amino, carboxyl,sulfhydryl, and aldehyde. There are many permutations of appropriatemoieties which will react with these groups and with differentlyformulated structures, to conjugate them together. See the PierceCatalog, and Merrifield, R. B., et al. Ciba Found Symp. 186:5-20, 1994.

The production and purification of photosensitizer:targeting moietyconjugates can be practiced by methods known in the art. Yield fromcoupling reactions can be assessed by spectroscopy of product elutingfrom a chromatographic fractionation in the final step of purification.The presence of uncoupled photosensitizer and reaction productscontaining the photosensitizer can be followed by the physical propertythat the photosensitizer moiety absorbs light at a characteristicwavelength and extinction coefficient, so incorporation into productscan be monitored by absorbance at that wavelength or a similarwavelength. Coupling of one or more photosensitizer molecules to atargeting moiety or to a backbone shifts the peak of absorbance in theelution profile in fractions eluted using sizing gel chromatography,e.g., with the appropriate choice of Sephadex G50, G100, or G200 orother such matrices (Pharmacia-Biotech, Piscataway N.J.). Choice ofappropriate sizing gel, for example Sephadex gel, can be determined bythat gel in which the photosensitizer elutes in a fraction beyond theexcluded volume of material too large to interact with the bead, i.e.,the uncoupled starting photosensitizer composition interacts to someextent with the fractionation bead and is concomitantly retarded to someextent. The correct useful gel can be predicted be predicted from themolecular weight of the uncoupled photosensitizer. The successfulreaction products of photosensitizer compositions coupled to additionalmoieties generally have characteristic higher molecular weights, causingthem to interact with the chromatographic bead to a lesser extent, andthus appear in fractions eluting earlier than fractions containing theuncoupled photosensitizer substrate. Unreacted photosensitizer substrategenerally appears in fractions characteristic of the starting material,and the yield from each reaction can thus be assessed both from size ofthe peak of larger molecular weight material, and the decrease in thepeak of characteristic starting material. The area under the peak of theproduct fractions is converted to the size of the yield using the molarextinction coefficient.

The product can be analyzed using NMR, integrating areas of appropriateproduct peaks, to determine relative yields with different couplingagents. A red shift in absorption of a photosensitizer of several nm hasoften been observed following coupling to a polyamino acid. Coupling toa larger moiety such as a protein might produces a comparable shift, ascoupling to an antibody resulted in a shift of about 3-5 nm in thatdirection compared to absorption of the free photosensitizer. Relevantabsorption maxima and extinction coefficients in 0.1M NaOH/1% SDS are,for chlorin e6, 400 nm and 150,000 M⁻¹, cm⁻¹, and for benzoporphyrinderivative, 430 nm and 61,000 M⁻¹, cm⁻¹.

Backbone Moieties

Photosensitizer:targeting moiety conjugates of the invention includethose in which a photosensitizer is coupled directly to a targetingmoiety, such as a histatin. Other photosensitizer:targeting moietyconjugates of the invention include a “backbone” or “bridge” moiety,such as a polyamino acid, which backbone is coupled both to aphotosensitizer and to a targeting moiety. The backbone can itself be atargeting moiety, e.g. polylysine (see Example 4 and FIGS. 5 and 6).

Inclusion of a backbone in a conjugate with a photosensitizer moiety anda targeting moiety can provide a number of advantages, including theprovision of greater stoichiometric ranges of photosensitizer andtargeting moieties coupled per backbone. If the backbone possessesintrinsic affinity for a target organism, the affinity of thecomposition can be enhanced by coupling to the backbone. The specificrange of organisms that can be targeted with one composition can beexpanded by coupling two or more different targeting moieties to asingle photosensitizer-backbone composition.

Peptides useful in the methods and compounds of the invention for designand characterization of backbone moieties include poly-amino acids whichcan be homo- and hetero-polymers of L-, D-, racemic DL- or mixed L- andD-amino acid composition, and which can be of defined or random mixedcomposition and sequence. Examples of naturally-occurring peptides withmixed D and L amino acid residues include bacitracin and tyrocidin.These peptides may be modeled after particular natural peptides, andoptimized by the technique of phage display and selection for enhancedbinding to a chosen target, so that the selected peptide of highestaffinity is characterized and then produced synthetically. Furthermodifications of functional groups can be introduced for purposes, forexample, of increased solubility, decreased aggregation, and alteredextent of hydrophobicity. Examples of nonpeptide backbones includenucleic acids and derivatives of nucleic acids such as DNA, RNA andpeptide nucleic acids; polysaccharides and derivatives such as starch,pectin, chitins, celluloses and hemi-methylated celluloses; lipids suchas triglyceride derivatives and cerebrosides; synthetic polymers such aspolyethylene glycols (PEGs) and PEG star polymers; dextran derivatives,polyvinyl alcohols, N-(2-hydroxypropyl)-methacrylamide copolymers, poly(DL-glycolic acid-lactic acid); and compositions containing elements ofany of these classes of compounds.

Modification of the Charge of Conjugates

The affinity of a conjugate for a target organism can be refined bymodifying the charge of a component of the conjugate.

Conjugates such as poly-L-lysine chlorin e6 can be made in varying sizesand charges (cationic, neutral, and anionic), for example, free NH₂groups of the polylysine are capped with acetyl, succinyl, or other Rgroups to alter the charge of the final composition. Net charge of aconjugate of the present invention can be determined by isoelectricfocusing (IEF). This technique uses applied voltage to generate a pHgradient in a non-sieving acrylamide or agarose gel by the use of asystem of ampholytes (synthetic buffering components). When chargedpolypeptides are applied to the gel they will migrate either to higherpH or to lower pH regions of the gel according to the position at whichthey become non-charged and hence unable to move further. This positioncan be determined by reference to the positions of a series of known IEFmarker proteins.

Due to the combination of polar charged groups on the polyaminoacid, andthe hydrophobic attraction between the planar aromatic tetrapyrrolerings, these conjugates can adopt pH dependent conformations which caninteract with bacterial cell walls. In addition, histatins and relatedpolypeptides contain at least one lysine residue which by theapplication of two heterobifunctional reagents will lead to a covalentdisulfide link between the histatin and the polylysine chlorin e6molecules. The optimum composition, concentration and time ofapplication of the photosensitizer to various pathogenic oral bacteriacan be determined.

Targeting Moieties

Desirable characteristics for the targeting moieties include:specificity for one or more unwanted target organisms, affinity andavidity for such organisms, and stability with respect to conditions ofcoupling reactions and the physiology of the organ or tissue of use.Specificity need not be narrowly defined, e.g., it may be desirable fora targeting molecule to have affinity for a broad range of targetorganisms, such as all Gram negative bacteria.

The targeting moiety, when incorporated into a conjugate molecule of theinvention, should be nontoxic to the cells of the subject.

Targeting moieties can be selected from the sequences of naturallyoccurring proteins and peptides, from variants of these peptides, andfrom biologically or chemically synthesized peptides. Naturallyoccurring peptides which have affinity for one or more target organismcan provide sequences from which additional peptides with desiredproperties, e.g., increased affinity or specificity, can be synthesizedindividually or as members of a library of related peptides. Suchpeptides can be selected on the basis of affinity for the targetorganism.

Naturally occurring peptides with affinity for target organisms usefulin methods and compounds of the invention, include salivary proteins,e.g., histatins, microbially-elaborated proteins, e.g., bacteriocins,peptides that bind and/or kill species that are closely related to theproducing strains; and proteins produced by animal species such asdefensins, which are produced by mammals, and the cecropins andmagainins, produced by moths and amphibia, respectively.

Histatins, defensins, cecropins and magainins are examples of a class ofpolypeptides found widely in nature, which share the characteristics ofsmall size (generally approximately 30 amino acid residues, and between10 residues and 50 residues), broad specificity of anti-microbialactivity, and low affinity for target organisms.

The use of histatins as a photosensitizer targeting moieties will allowtargeting a photosensitizer to a bacterial cell while leaving the hosttissue unharmed. Histatins are a family of histidine-rich cationicpolypeptides which have bactericidal and candidacidal properties and areconstituents of normal human saliva (Oppenheim, G. G. et al., J. Biol.chem. 263:7472-747, 1988). Their mechanism of action is thought toinvolve a combination of alpha-helical conformation and cationic chargeleading them to insert between the polar head groups in the bacterialcell wall (Raj, P. A. et al., J. Biol. Chem. 269:9610-9619, 1994).

While histatins can be used usefully employed as oral bacteriocides,their action occurs over time periods of hours, leading to the problemof formulating delivery vehicles such as gels to keep the histatins inthe region of infection. Photodynamic inactivation of oral bacteria,however, can require only brief application of the bacteria-targetedphotosensitizer, such as by supplying in a mouthwash. Because bacteriaare 50-100 times smaller than the average mammalian cell and themechanism of photodynamic therapy is thought to involve the productionof molecular species such as singlet oxygen which have very shortdiffusion distances in tissues (less than 50 nm for singlet oxygen), itcan be seen that modest levels of sensitizer selectivity for bacteriamay lead to high levels of selectivity in cytotoxicity. Low levels ofPDT in humans and experimental animals have been shown to activatecomponents of the host immune system such as macrophages andlymphocytes, and these activated host cells may play a part indestroying bacteria and helping the regeneration of tissue destroyed bydisease.

Histatins-1, -3 and -5 each contain 7 residues of histidine, in a totalpolypeptide length of 38, 32 and 24 residues, respectively. Histatinshave a number of activities, for example, an anti-fungal activity, forexample, against Candida pathogens. (U.S. Pat. No. 5,486,503).Recombinant duplication of histatin-5 residues 13-24 gives a peptidewith enhanced candidacidal activity (Zuo, F. et al., Gene 161:87-91,1995). Histatin-5 is an inhibitor of the trypsin-like protease producedby the oral bacterial species Porphyromonas (Bacteroides) gingivalis,which protease is associated with tissue destruction of periodontaldisease (Nishikata, M. et al., Biochem. Biophys. Res. Comm. 174:625-630,1991). About 3,600 histatin-5 molecules bind P. gingivalis with a K_(d)on the order of 10⁻⁶ M (Murakami, Y. et al., FEMS Microbiol. Letts.82:253-256,1991). Histatins-5 and -8 inhibit coaggregation of P.gingivalis and S. mitis (Murakami, Y. et al., Inf. Immun. 59:3284-3286,1991), which may modulate the attachment of P. gingivalis to Grampositive bacteria previously bound to oral tissues.

Histatin-5 has bactericidal activity against at least the oral bacterialspecies P. gingivalis (Colon, J. O. et al., J. Dent. Res. 72 IADRAbstr.:322, Abstr. 1751) and Actinomyces viscosus, A. naeslundii, and A.odontolyticus (Kalpidis, C. D. et al., op. cit. 71:305, Abstr. 1595).The direct anti-microbial activity against the latter species appears tobe without receptor activity for agglutination of Actinomyces cells. Asynthetic peptide of histatin-5 is a potent inhibitor of P. (B.)gingivalis hemagglutinin (Murakami, Y. et al., Archs. Oral. Biol.35:775-777). The synthetic peptide is strongly cationic (containing 6His, 4 Lys, and 3 Arg in 22 residues) and may function as the bindingdomain for P. gingivalis on epithelial cells, salivary pellicle, andGram positive cells.

Histatins that have been chemically capped at the C- or N-terminus, andcomplexed with a metal, for example Ag, Cu, Zn or Sn, are suitable for arange of anti-microbial applications, such as antiplaque, anti-caries,anti-bad breath oral applications, deodorant applications, personalhygiene applications and so on (EPO Patent Application 721 774 A2).

Bacteriocins, which are proteins produced by bacteria and which killother strains and species of bacteria (Jack, R. W. et al., Microbiol.Rev. 59:171-200, 1995) can be used as targeting moieties. An exemplaryGram positive bacteriocin is nisin, produced by Lactococcus lactis andaccorded GRAS status (generally regarded as safe) by the Food and DrugAdministration for application to food preservation.

The bacteriocins nisin, subtilin, epidermin, gallidermin, salivarin, andlacticin exemplify the “lantibiotic” class of Gram positive bacteriocin,which is defined as a bacteriocin in which one or more cysteine residuesare linked to a dehydrated serine or threonine to form athioether-linked residue known as lanthionine (Lan) orthreo-β-methyllanthionine (MeLan). These are post-translationalmodifications found in these anti-microbial peptides by the producingcell. Lantibiotics contain leader peptide sequences of 18-24 residues,which are cleaved to yield an active antimicrobial peptide of about22-35 residues. Growth of the producing bacterial species, andpreparation and purification of bacteriocins are performed by publishedprocedures and techniques which can be carried out by one of skill inthe art. For example, Yang, R. et al., Appl. and Env. Microbiol 58:3355-3359, 1992, describe purification of bacteriocins from each of 4genera of lactic acid bacteria, by optimizing absorption onto theproducing cells, followed by use of low pH for selective elution ofgreatly enriched bacteriocin fractions. Mutant forms of each of thebacteriocins nisin, produced by Lactococcus lactis, and of subtilin,produced by Bacillus subtilis have more desirable properties than theparental wild-type forms (Liu, W. and N. Hansen, J. Biol. Chem.267:25,078-25,085, 1992). Procedures for isolation of appropriate genesand for mutagenesis and selection of strains carrying desirablemutations are found in Maniatis, T. et al, 1982, Molecular Cloning: aLaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,and in the subsequent second edition, Sambrook, J. et al., 1989.

Anti-microbial peptides are produced by a variety of animals (see reviewby Saberwal, G. and R. Nagaraj, Biochim. Biophys. Act. 1197:109-131,1994). An example is a peptide of the cecropin family produced byCecropia moths. Several cecropins contain 37 residues, of which 6 arelysine. Cecropins are active against both Gram positive and Gramnegative bacteria. Other insect produced peptides include apidaecin(from honeybees), andropin (from fruit flies), and cecropin familymembers from bumble bees, fruit flies, and other insects.

The defensins are produced by mammals, including humans, and aregenerally about 29-34 residues in length, and the magainins (about 23residues) are produced by amphibia such as Xenopus laevis. Defensinsfrom human (HNP-1,-2,-3 and 4), guinea pig (GPNP), rabbit (NP-1, -2,-3A, -3B, -4 and -5) and rat (NP-1, -2, -3 and -4) share a significantnumber of regions of homology. Defensins can have antimicrobial activityagainst Gram positive bacteria or Gram negative bacteria and fungi, withminimal inhibitory concentrations in the mM range. Rabbit NP-1 and NP-2are more potent antibacterial agents than others in this family. Othermammalian anti-microbial peptides include murine cryptdin, bovinegranulocyte bactenecin and indolicidin, and seminal-plasmin from bovinesemen. Additional amphibial anti-microbials include PGLA, XPF, LPF, CPG,PGQ, bombinin from Bombina variegata, the bombinin-like peptides BLP-1,-2, -3 and -4 from B. orientalis, and brevinins from Rana esculenta.Invertebrates such as the horseshoe crab can be a source ofanti-microbial peptides such as the tachyplesins (I, II and III) and thepolyphemusins (I and II).

Peptides in these families of antimicrobial agents are generallycationic, and can have a broad antimicrobial spectrum, including bothantibacterial and antifungal activities. The addition of positivelycharged residues can enhance antimicrobial specific activity severalfold. The positive charges are thought to assist in the insertion of thepeptides into the membranes of the susceptible organisms, in whichcontext the peptide molecules can form pores and cause efflux of ionsand other metabolites. Structural studies of the Moses sole fishneurotoxin 33 residue peptide pardaxin, for example, reveals thatsuccinylated pardaxin inserts into erythrocyte and model membranes moreslowly than unmodified pardaxin. (Shai, Y et al., J. Biol. Chem. 265:20, 202-20, 209, 1990). The positively charged magainin molecule candisrupt both the metabolism of E. coli and the electric potential of themitochondrion (Westerhoff, H. V., et al., Proc. Natl. Acad. Sci.86:6597-6601, 1989).

Novel peptides, for example a cecropin-melittin hybrid, and syntheticDenantiomers, have antimicrobial activity (Merrifield, R. B. et al.,“Antimicrobial peptides,” Ciba Foundation Symp. 186, John Wiley,Chichester, pp. 5-26, 1994). One such synthetic cecropin-melittinpeptide is 5-fold more active against Mycobacterium smegmatis thanrifampin.

Targeting moieties can be plant proteins with affinities for particulartarget organisms, for example, a member of the lectin protein familywith affinity for polysaccharides.

Targeting moieties can be synthetic peptides, such as polylysine,polyarginine, polyornithine, and synthetic heteropolypeptides thatcomprise substantial proportions of such positively charged amino acidresidues. Such peptides can be chemically synthesized or producedbiologically in recombinant organisms, in which case the targetingmoiety peptide can be produced as part of a larger protein, for exampleas the N-terminus residues, and cleaved from that larger protein.Polypeptides suitable as “backbone” and “bridge” moieties are alsosuitable as target moieties, if they have sufficient affinity for thetarget organism. Considerations described are thus appropriate toconsideration of a targeting moieties. Targeting moieties can besynthesized and selected or enriched by the variety of methods describedherein.

Targeting moieties need not be limited to peptide compositions, but canbe lectins, polysaccharides, steroids, and metalloorganic compositions.Targeting moieties can be comprised of compositions that are composedboth of amino acids and sugars, such as mucopolysaccharides. A usefultargeting moiety can be partially lipid and partially peptide in nature,such as low density lipoprotein. Serum lipoproteins especially highdensity and low density lipoproteins (HDL and LDL) can bind to bacterialsurface proteins (Emancipator, K. et al., Infect. Immun. 60:596-601,1992). HDL and especially reconstituted HDL neutralizes bacteriallipopolysaccharide both in vitro and in vivo (Wurfel MM et al., J. Exp.Med. 181:1743—1754, 1995). Endogenous LDL can protect against the lethaleffects of endotoxin and Gram negative infection (Netea, M., et al., J.Clin. Invest. 97:1366-1372, 1996). The appropriate binding features ofthe lipoproteins to bacterial surface components can be identified bymethods of molecular biology known in the art, and the binding featureof lipoproteins can be used as the targeting moiety in photosensitizercompositions of the present invention.

Production and Screening of Peptide Targeting Moiety Candidates

The inventor has discovered that molecules, e.g., peptides, other thanantibodies and members of a high affinity ligand pairs, can be used totarget a photosensitizer to a target organism. The following methods canbe used to modify or refine the targeting moieties disclosed herein orto discover new targeting moieties.

Once an example of a targeting moiety of reasonable affinity has beenprovided, one skilled in the art can alter the disclosed structure (of apolylysine polypeptide, for example), by producing fragments or analogs,and testing the newly produced structures for modification of affinityor specificity. Examples of methods which allow the production andtesting of fragments and analogs are discussed below. These methods canbe used to make fragments and analogs of a known naturally occurringpolypeptide or protein which is a targeting moiety, e.g., a polypeptidesuch as histatin or low density lipoprotein, each of which has bindingaffinity for cells of one or more bacterial species.

Generation of Fragments

Fragments of a protein can be produced in several ways, e.g.,recombinantly, by proteolytic digestion, or by chemical synthesis.Internal or terminal fragments of a polypeptide can be generated byremoving one or more nucleotides from one end (for a terminal fragment)or both ends (for an internal fragment) of a nucleic acid which encodesthe polypeptide. Expression of the mutagenized DNA produces polypeptidefragments. Digestion with “end-nibbling” processive exonucleases canthus generate DNA's which encode an array of fragments. DNA's whichencode fragments of a protein can also be generated by random shearing,restriction digestion or a combination of the above-discussed methods.

Fragments can also be chemically synthesized using techniques known inthe art such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. For example, peptides of the present invention may bearbitrarily divided into fragments of desired length with no overlap ofthe fragments, or divided into overlapping fragments of a desiredlength.

Generation of Analogs: Production of Altered DNA and Peptide Sequencesby Random Methods

Amino acid sequence variants of a protein can be prepared by randommutagenesis of DNA which encodes a protein or a particular domain orregion of a protein. Useful methods include PCR mutagenesis andsaturation mutagenesis. A library of random amino acid sequence variantscan also be generated by the synthesis of a set of degenerateoligonucleotide sequences. (Methods for screening proteins in a libraryof variants are elsewhere herein.)

PCR Mutagenesis

In PCR mutagenesis, reduced Taq polymerase fidelity is used to introducerandom mutations into a cloned fragment of DNA (Leung et al., 1989,Technique 1:11-15). This is a very powerful and relatively rapid methodof introducing random mutations. The DNA region to be mutagenized isamplified using the polymerase chain reaction (PCR) under conditionsthat reduce the fidelity of DNA synthesis by Taq DNA polymerase, e.g.,by using a dGTP/dATP ratio of five and adding Mn²⁺ to the PCR reaction.The pool of amplified DNA fragments are inserted into appropriatecloning vectors to provide random mutant libraries.

Saturation Mutagenesis

Saturation mutagenesis allows for the rapid introduction of a largenumber of single base substitutions into cloned DNA fragments (Mayers etal., 1985, Science 229:242). This technique includes generation ofmutations, e.g., by chemical treatment or irradiation of single-strandedDNA in vitro, and synthesis of a complementary DNA strand. The mutationfrequency can be modulated by modulating the severity of the treatment,and essentially all possible base substitutions can be obtained. Becausethis procedure does not involve a genetic selection for mutant fragmentsboth neutral substitutions, as well as those that alter function, areobtained. The distribution of point mutations is not biased towardconserved sequence elements.

Degenerate Oligonucleotides

A library of homologs can also be generated from a set of degenerateoligonucleotide sequences. Chemical synthesis of a degenerate sequencescan be carried out in an automatic DNA synthesizer, and the syntheticgenes then ligated into an appropriate expression vector. The synthesisof degenerate oligonucleotides is known in the art (see for example,Narang, S A (1983) Tetrahedron 39:3; Itakura et al. (1981) RecombinantDNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. A G Walton,Amsterdam: Elsevier pp 273-289; Itakura et al. (1984) Annu. Rev.Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.(1983) Nucleic Acid Res. 11:477. Such techniques have been employed inthe directed evolution of other proteins (see, for example, Scott et al.(1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433;Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

Generation of Analogs: Production of Altered DNA and Peptide Sequencesby Directed Mutagenesis

Non-random or directed, mutagenesis techniques can be used to providespecific sequences or mutations in specific regions. These techniquescan be used to create variants which include, e.g., deletions,insertions, or substitutions, of residues of the known amino acidsequence of a protein. The sites for mutation can be modifiedindividually or in series, e.g., by (1) substituting first withconserved amino acids and then with more radical choices depending uponresults achieved, (2) deleting the target residue, or (3) insertingresidues of the same or a different class adjacent to the located site,or combinations of options 1-3.

Alanine Scanning Mutagenesis

Alanine scanning mutagenesis is a useful method for identification ofcertain residues or regions of the desired protein that are preferredlocations or domains for mutagenesis, Cunningham and Wells (Science244:1081-1085, 1989). In alanine scanning, a residue or group of targetresidues are identified (e.g., charged residues such as Arg, Asp, His,Lys, and Glu) and replaced by a neutral or negatively charged amino acid(most preferably alanine or polyalanine). Replacement of an amino acidcan affect the interaction of the amino acids with the surroundingaqueous environment in or outside the cell. Those domains demonstratingfunctional sensitivity to the substitutions are then refined byintroducing further or other variants at or for the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to optimize the performance of amutation at a given site, alanine scanning or random mutagenesis may beconducted at the target codon or region and the expressed desiredprotein subunit variants are screened for the optimal combination ofdesired activity.

Oligonucleotide-Mediated Mutagenesis

Oligonucleotide-mediated mutagenesis is a useful method for preparingsubstitution, deletion, and insertion variants of DNA, see, e.g.,Adelman et al., (DNA 2:183, 1983). Briefly, the desired DNA is alteredby hybridizing an oligonucleotide encoding a mutation to a DNA template,where the template is the single-stranded form of a plasmid orbacteriophage containing the unaltered or wild type DNA sequence of thedesired protein. After hybridization, a DNA polymerase is used tosynthesize an entire second complementary strand of the template thatwill thus incorporate the oligonucleotide primer, and will code for theselected alteration in the desired protein DNA. Generally,oligonucleotides of at least 25 nucleotides in length are used. Anoptimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al. (Proc.Natl. Acad. Sci. USA, 75: 5765, 1978).

Cassette Mutagenesis

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al. (Gene, 34:315, 1985). Thestarting material is a plasmid (or other vector) which includes theprotein subunit DNA to be mutated. The codon(s) in the protein subunitDNA to be mutated are identified. There must be a unique restrictionendonuclease site on each side of the identified mutation site(s). If nosuch restriction sites exist, they may be generated using theabove-described oligonucleotide-mediated mutagenesis method to introducethem at appropriate locations in the desired protein subunit DNA. Afterthe restriction sites have been introduced into the plasmid, the plasmidis cut at these sites to linearize it. A double-stranded oligonucleotideencoding the sequence of the DNA between the restriction sites butcontaining the desired mutation(s) is synthesized using standardprocedures. The two strands are synthesized separately and thenhybridized together using standard techniques. This double-strandedoligonucleotide is referred to as the cassette. This cassette isdesigned to have 3′ and 5′ ends that are comparable with the ends of thelinearized plasmid, such that it can be directly ligated to the plasmid.This plasmid now contains the mutated desired protein subunit DNAsequence.

Combinatorial Mutagenesis

Combinatorial mutagenesis can also be used to generate mutants. E.g.,the amino acid sequences for a group of homologs or other relatedproteins are aligned, preferably to promote the highest homologypossible. All of the amino acids which appear at a given position of thealigned sequences can be selected to create a degenerate set ofcombinatorial sequences. The variegated library of variants is generatedby combinatorial mutagenesis at the nucleic acid level, and is encodedby a variegated gene library. For example, a mixture of syntheticoligonucleotides can be enzymatically ligated into gene sequences suchthat the degenerate set of potential sequences are expressible asindividual peptides, or alternatively, as a set of larger fusionproteins containing the set of degenerate sequences.

Primary High-Throughput Methods for Screening Libraries of PeptideFragments or Homologs

Various techniques are known in the art for screening generated geneproducts. Techniques for screening large libraries often include cloningthe nucleic acids of interest into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the genes under conditions in which detection of adesired activity, e.g., in this case, binding to a target organism or asurface component of a target organism, facilitates relatively easyisolation of the vector encoding the gene whose product was detected.Each of the techniques described below is amenable to high through-putanalysis for screening large numbers of sequences created, e.g., byrandom mutagenesis techniques.

Display Libraries

In one approach to screening assays, the candidate peptides aredisplayed on the surface of a cell or viral particle, and the ability ofparticular cells or viral particles to bind an appropriate targetorganisms protein via the displayed product is detected in a “panningassay”. For example, the gene library can be cloned into the gene for asurface membrane protein of a bacterial cell, and the resulting fusionprotein detected by panning (Ladner et al., WO 88/06630; Fuchs et al.(1991) Bio/Technology 9:1370-1371; and Goward et al. (1992) TIBS18:136-140). In a similar fashion, a detectably labeled ligand can beused to score for potentially functional peptide homologs. Fluorescentlylabeled ligands, e.g., target organisms, can be used to detect homologswhich retain ligand-binding activity. The use of fluorescently labeledligands, allows cells to be visually inspected and separated under afluorescence microscope, or, where the morphology of the cell permits,to be separated by a fluorescence-activated cell sorter.

A gene library can be expressed as a fusion protein on the surface of aviral particle. For instance, in the filamentous phage system, foreignpeptide sequences can be expressed on the surface of infectious phage,thereby conferring two significant benefits. First, since these phagecan be applied to affinity matrices at concentrations well over 10¹³phage per milliliter, a large number of phage can be screened at onetime. Second, since each infectious phage displays a gene product on itssurface, if a particular phage is recovered from an affinity matrix inlow yield, the phage can be amplified by another round of infection. Thegroup of almost identical E. coli filamentous phages M13, fd, and fl aremost often used in phage display libraries. Either of the phage gIII orgVIII coat proteins can be used to generate fusion proteins withoutdisrupting the ultimate packaging of the viral particle. Foreignepitopes can be expressed at the NH₂-terminal end of pIII and phagebearing such epitopes recovered from a large excess of phage lackingthis epitope (Ladner et al. PCT publication WO 90/02909; Garrard et al.,PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem.267:16007-16010; Griffiths et al. (1993) EMBO J 12:725-734; Clackson etal. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS89:4457-4461).

A common approach uses the maltose receptor of E. coli (the outermembrane protein, LamB) as a peptide fusion partner (Charbit et al.(1986) EMBO 5, 3029-3037). Oligonucleotides have been inserted intoplasmids encoding the LamB gene to produce peptides fused into one ofthe extracellular loophotosensitizer of the protein. These peptides areavailable for binding to ligands, e.g., to antibodies, and can elicit animmune response when the cells are administered to animals. Other cellsurface proteins, e.g., OmpA (Schorr et al. (1991) Vaccines 91, pp.387-392), PhoE (Agterberg, et al. (1990) Gene 88, 37-45), and PAL (Fuchset al. (1991) Bio/Tech 9, 1369-1372), as well as large bacterial surfacestructures have served as vehicles for peptide display. Peptides can befused to pilin, a protein which polymerizes to form the pilus-a conduitfor interbacterial exchange of genetic information (Thiry et al. (1989)Appl. Environ. Microbiol. 55, 984-993). Because of its role ininteracting with other cells, the pilus provides a useful support forthe presentation of peptides to the extracellular environment. Anotherlarge surface structure used for peptide display is the bacterial motiveorgan, the flagellum. Fusion of peptides to the subunit proteinflagellin offers a dense array of may peptides copies on the host cells(Kuwajima et al. (1988) Bio/Tech. 6, 1080-1083). Surface proteins ofother bacterial species have also served as peptide fusion partners.Examples include the Staphylococcus protein A and the outer membrane IgAprotease of Neisseria (Hansson et al. (1992) J. Bacteriol. 174,4239-4245; Klauser et al. (1990) EMBO J. 9, 1991-1999).

In the filamentous phage systems and the LamB system described above,the physical link between the peptide and its encoding DNA occurs by thecontainment of the DNA within a particle (cell or phage) that carriesthe peptide on its surface. Capturing the peptide captures the particleand the DNA within. An alternative scheme uses the DNA-binding proteinLacl to form a link between peptide and DNA (Cull et al., 1992, PNAS USA89:1865-1869). This system uses a plasmid containing the LacI gene withan oligonucleotide cloning site at its 3′-end. Under the controlledinduction by arabinose, a LacI-peptide fusion protein is produced. Thisfusion retains the natural ability of Lacl to bind to a short DNAsequence known as LacO operator (LacO). By installing two copies of LacOon the expression plasmid, the LacI-peptide fusion binds tightly to theplasmid that encoded it. Because the plasmids in each cell contain onlya single oligonucleotide sequence and each cell expresses only a singlepeptide sequence, the peptides become specifically and stably associatedwith the DNA sequence that directed its synthesis. The cells of thelibrary are gently lysed and the peptide-DNA complexes are exposed to amatrix of immobilized receptor to recover the complexes containingactive peptides. The associated plasmid DNA is then reintroduced intocells for amplification and DNA sequencing to determine the identity ofthe peptide ligands. As a demonstration of the practical utility of themethod, a large random library of dodecapeptides was made and selectedon a monoclonal antibody raised against the opioid peptide dynorphin B.A cohort of peptides was recovered, all related by a consensus sequencecorresponding to a six-residue portion of dynorphin B (Cull et al.(1992) Proc. Natl. Acad. Sci. U.S.A. 89-1869).

This scheme, sometimes referred to as peptides-on-plasmids, differs intwo important ways from the phage display methods. First, the peptidesare attached to the C-terminus of the fusion protein, resulting in thedisplay of the library members as peptides having free carboxy termini.Both of the filamentous phage coat proteins, pIII and pVIII, areanchored to the phage through their C-termini, and the guest peptidesare placed into the outward-extending N-terminal domains. In somedesigns, the phage-displayed peptides are presented right at the aminoterminus of the fusion protein. (Cwirla, et al. (1990) Proc. Natl. Acad.Sci. U.S.A. 87, 6378-6382) A second difference is the set of biologicalbiases affecting the population of peptides actually present in thelibraries. The Lacl fusion molecules are confined to the cytoplasm ofthe host cells. The phage coat fusions are exposed briefly to thecytoplasm during translation but are rapidly secreted through the innermembrane into the periplasmic compartment, remaining anchored in themembrane by their C-terminal hydrophobic domains, with the N-termini,containing the peptides, protruding into the periplasm while awaitingassembly into phage particles. The peptides in the LacI and phagelibraries may differ significantly as a result of their exposure todifferent proteolytic activities. The phage coat proteins requiretransport across the inner membrane and signal peptidase processing as aprelude to incorporation into phage. Certain peptides exert adeleterious effect on these processes and are underrepresented in thelibraries (Gallop et al. (1994) J. Med. Chem. 37(9):1233-1251). Theseparticular biases are not a factor in the LacI display system.

The number of small peptides available in recombinant random librariesis enormous. Libraries of 10⁷-10⁹ independent clones are routinelyprepared. Libraries as large as 10¹¹ recombinants have been created, butthis size approaches the practical limit for clone libraries. Thislimitation in library size occurs at the step of transforming the DNAcontaining randomized segments into the host bacterial cells. Tocircumvent this limitation, an in vitro system based on the display ofnascent peptides in polysome complexes has recently been developed. Thisdisplay library method has the potential of producing libraries 3-6orders of magnitude larger than the currently available phage/phagemidor plasmid libraries. Furthermore, the construction of the libraries,expression of the peptides, and screening, is done in an entirelycell-free format.

In one application of this method (Gallop et al. (1994) J. Med. Chem.37(9):1233-1251), a molecular DNA library encoding 10¹² decapeptides wasconstructed and the library expressed in an E. coli S30 in vitro coupledtranscription/translation system. Conditions were chosen to stall theribosomes on the mRNA, causing the accumulation of a substantialproportion of the RNA in polysomes and yielding complexes containingnascent peptides still linked to their encoding RNA. The polysomes aresufficiently robust to be affinity purified on immobilized receptors inmuch the same way as the more conventional recombinant peptide displaylibraries are screened. RNA from the bound complexes is recovered,converted to cDNA, and amplified by PCR to produce a template for thenext round of synthesis and screening. The polysome display method canbe coupled to the phage display system. Following several rounds ofscreening, cDNA from the enriched pool of polysomes was cloned into aphagemid vector. This vector serves as both a peptide expression vector,displaying peptides fused to the coat proteins, and as a DNA sequencingvector for peptide identification. By expressing the polysome-derivedpeptides on phage, one can either continue the affinity selectionprocedure in this format or assay the peptides on individual clones forbinding activity in a phage ELISA, or for binding specificity in acompletion phage ELISA (Barret, et al. (1992) Anal. Biochem204,357-364). To identify the sequences of the active peptides onesequences the DNA produced by the phagemid host.

Secondary Screens

The high through-put assays described above can be followed by secondaryscreens in order to identify, enrich and select for molecules havingappropriate affinity for a biological entity. Secondary screens dependon the ability of the targeting moiety to bind a polymer of interest.For example, a surface protein or carbohydrate of the target organism ofinterest can be used to identify ligands from a group of peptidefragments isolated though one of the primary screens described above.One may use highly pure materials, for example, purified protein from aviral pathogen, obtained from a recombinant organism specificallyobtained for the purpose of production of this material, or one may usea crude preparation of the target organism, such as a cell-wall orpellicle preparation, even a heat-inactivated or formalin-treatedpreparation of the target organism.

The Examples below illustrate two examples of targeting materials, apolyamino acid of positive charge, polylysine, which has affinity for abroad range of bacterial species and can also serve as a backbone forcoupling of additional targeting moieties; and the salivary proteinhistatin, which has affinity for several species of oral bacteria. Eachof these materials can be used as a starting material in the proceduresdescribed for phage display library, described herein, for example byincorporation of the nucleic acid sequence into that of gene III of theM13 phage display vector (see, for example, Scott et al. (1990) Science249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al.(1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; aswell as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815). In thiscase, the target material for enrichment of the phage library may bebacterial cell wall fraction, isolated by sonication of the targetorganism in the cold in the presence of standard protease inhibitors,low speed centrifugation to separate cell walls from cytoplasm, andresuspension of wall material in buffer for use in several rounds ofphage library binding selection. Procedures are described at length inU.S. Pat. No. 5,223,409. After two to three rounds of selection, phagebearing polyamino acid sequences or histatin variants of affinity to thetarget cell wall that is considerably enhanced over that of the startingmaterial may be isolated. The sequence of the improved variants isreadily determined by standard DNA sequence procedures, and the peptidecan be produced in large quantity by standard peptide synthesis methods.Thus the procedures described here for generating fragments and analogsand testing them for enhanced affinity for the target organism are knownin the art.

Target Organisms

Organisms to be targeted by the compositions and methods of the presentinvention are found on any light-accessible surfaces or inlight-accessible areas, e.g., in human and animal subjects, on materialsto be decontaminated, or on crop plants. In the cases of humans andanimals, infections of the epidermis, oral cavity, nasal cavity,sinuses, ears, lungs, urogenital tract, and gastrointestinal tract arelight accessible. Epidermal infections include those of unwantedorganisms of bacterial, fungal, viral and animal origin, and includesubcutaneous infections, especially localized lesions, for examplecaused by protozoans, parasites, or parasitic mites, which infectionsare light-accessible. Infections of the peritoneal cavity, such as thoseresulting from burst appendicitis, are light accessible via at leastlaparoscopic devices. A variety of skin infections which are refractoryto antibiotics or long-term antifungal treatment, for example,dermatophycoses of the toenail, are suitable for PDT using thecompositions of the invention.

A major area of application of compositions and methods of the inventionare disorders and infections of the oral cavity, e.g., of the gums.Methods of the invention are particularly useful in treating oralinfectious diseases, for example, periodontal diseases. Since pockets ofperiodontal disease infection occur within a few millimeters of thesurface of the oral cavity, PDT offers significant advantages over thetraditional physical methods of scaling and antibiotic therapy for thiscondition. Target oral unwanted organisms include a large number ofbacterial and fungal species, e.g., Bacteroides species including B.gingivalis (now known as Porphyromonas gingivalis), Eikenella corrodens,Fusobacterium nucleatum, Wolinella recta, Eubacterium species,Prevotella (Bacteroides) intermedia, Bacteroides forsythus,Capnocytophaga species, Actinobacillus actinomycetamcomitans, andStreptococcus mutans.

Lung infection can occur with a variety of bacterial genera and species,which include the classical tuberculosis of Mycobacterium tuberculosis,the pseudomonads, which are the primary cause of death of cysticfibrosis patients, Klebsiella, and can also occur with a variety ofvirus strains. A variety of fungi and parasites are opportunisticpathogens of the lung, and Pneumocystis carinii infection is a commoncause of death in immunocompromised AIDS patients. As pathogens of thelung are increasingly resistant to classical antibiotic therapies, PDTwith the compositions of the instant invention offer an alternativemethod for eliminating these unwanted organisms that is independent ofthe microbial mechanisms of resistance. Additional epidermal infectionsand infections of deeper tissues arise from burns, scrapes, cuts, andpuncture wounds. PDT with the compositions of the instant invention isuseful for sterilization of such potential infectious sites, which canrapidly lead to toxic shock, a frequent concomitant of bullet wounds,and for treating the sites to eliminate or reduce unwanted infectiousorganisms. A major cause of infection in wounds, especially burns, isthe Gram negative aerobic bacterium Pseudomonas. This organism producesan exotoxin which has been shown to retard wound healing.Multi-antibiotic resistant P. aeruginosa strains are becoming asignificant problem, especially in burns units of large hospitals.Pseudomonads also produce fulminating infections of the cornea.Escherichia coli along with Staphylococcus aureus are the two mostcommon bacteria in infected wounds.

Other sites of unwanted target organisms include the urogenital tract,the peritoneal cavity, the inner and outer ear, the nasal cavity and thegastrointestinal tract. Infectious sites of proliferation of unwantedtarget organisms in tissues of mesothelial and endothelial origin arealso accessible to PDT by minimally invasive techniques.

Target organisms can be cellular or viral. Viruses which can be unwantedtarget organisms include any pathogenic life form comprising componentsof at least one nucleic acid molecule and one or more protein species,and may also include the enveloped viruses. Target organisms which arecells include at least a boundary cell membrane and are capable ofenergy production, nucleic acid synthesis, and contain ribosomes and arecapable of ribosomal protein synthesis. Cells can be unicellular ormulticellular, and said unicellular organisms can be prokaryotic oreukaryotic.

Prokaryotic target organisms can be bacteria, which bacteria can be Gramnegative or Gram positive, or which are lacking cell walls. The Gramstain basis of distinguishing bacteria, based on whether or not cells ofa specific strain or species of bacteria take up a stain, or are stainedwith the counterstain only, is known to those of skill in the art.Bacteria which are target organisms of the invention can be aerobic,anaerobic, facultatively anaerobic or microaerophilic. Spirochetes ofthe invention include but are not limited to the genera Borrelia andTreponema. This last genus contains species variously associated withthe diseases of trenchmouth, pinta, and yaws, the latter two beingtropical skin infections. Gram negative helical/vibroid motile bacterialgenera suitable as target organisms include Campylobacter andHelicobacter. Gram negative aerobic and microaerophilic rods and cocciinclude the genera Bordetella, Neisseria, and Legionella. Facultativelyanaerobic Gram negative rods include genera Pseudomonas, Salmonella,Shigella, Erwinia, Enterobacter, Erwinia, Escherichia, Vibrio,Haemophilus, Actinobacillus, Klebsiella and Salmonella. An importantgroup of bacteria as target organisms for the present invention are theGram positive cocci, including the genera Staphylococcus andStreptococcus, a strain of the latter known to cause a variety ofinfections including the childhood skin disease impetigo, and somestrains of the former which are popularly designated, “flesh-eatingbacteria.” Gram positive rods include species of Listeria, suitable fortreatment by the methods and compositions of the invention.

Bacteria suitable for photosensitizer composition treatment among thoselacking rigid cell walls are the genus Mycoplasma. The actinomycetegroup includes several species of Mycobacterium that are suitable targetorganisms of the present invention. Additional bacterial genera whichcan be treated with the conjugate molecules of the invention include:Enterococcus, Leptospira, Serpulina, Mycoplasma, Bacteroides, Yersinia,Chlamydia, Vibrio, Actinobacillus, Porphyromonas, Hemophilus,Pasteurella, Peptostreptococcus, Propionibacterium, Corynebacterium andDermatophilus. These and other bacterial groups and genera not listedhere will be recognized by the skilled artisan as suitable targetbacteria for the compositions of the invention.

Viruses that may be targeted by the compositions of the presentinvention include, but are not limited to, adenoviruses, herpesviruses,poxviruses, and retroviruses. Representative fungal target organismgenera include but are not limited to, Cryptococcus, Blastomyces,Paracoccidioides, Candida, Aspergillus, Mycetoma, and include othergenera causing various dermatomycoses.

Eukaryotic target organisms of the instant invention include unicellularprotozoan and fungal pathogens and parasites, which can have amulticellular phase of the life cycle. Parasite infections of subjectsare suitable for treatment by the compositions of the invention. Commonparasites that infect or colonize the intestinal and urogenital tractinclude amoebae, flagellates, and nematodes. In addition, infection withtrematodes, cestodes, ciliates, coccidian and microsporidian parasitesmay occur in these tracts. Members of the genera Leishmania andOnchocerca cause cutaneous ulcers, and of the genus Acanthamoeba can befound in corneal scrapings of the eye. Leishmania donovani causes thetropical ulcerating skin disease kala azar, which is suitable fortreatment with the methods and compositions of the present invention.Intestinal tract genera that are suitable for targeting by compositionsof the invention include Entamoeba, Giardia, Cryptosporidium, andmicrosporidia, pinworm, and helminth genera. Lung tissue cancontainPneumocystis carinii, and more rarely, amoebae such as Entamoeba,trematodes, or cestodes. The urogenital tract can be infected withTrichomonas, and with Schistosoma, which can be treated withcompositions of the invention.

Viral, prokaryotic and eukaryotic target organisms are not limited topathogens and parasites, and can include higher orders such asarthropods. Target organisms are not limited to pathogens and parasitesof animal subjects, and can include plant pests.

These lists are used to illustrate applications of the present inventionto major groups of suitable target organisms, but not to delimit theinvention to the species, genera, families, orders or classes so listed.

Pharmaceutical Compositions

The compounds of the invention include conjugate molecules that havebeen formulated for topical administration, and also for administrationto various external organs such as the outer ear, or organs accessibleby external administration, such as by oral application or by lavage ofthe lung. The examples mentioned here are not intended as limiting withrespect to the nature of the conjugate photosensitizer compositions ofthe invention, or to a particular route of the administration, andadditional routes are listed herein. In another embodiment of thepresent invention, the photosensitizer compositions can be administeredby combination therapy, i.e., combined with other agents. For example,the combination therapy can include a composition of the presentinvention with at least one other photosensitizer, at least oneantibiotic, or other conventional therapy.

Photosensitizer conjugates that are somewhat insoluble in an aqueoussolvent can be applied in a liposome, or a time release fashion, suchthat illumination can be applied intermittently using a regimen ofperiods of illumination alternating with periods of non-illumination.Other regimens contemplated are continuous periods of lower levelillumination, for which a time-release formulation is suitable.

As used herein, the phrase “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike that are physiologically compatible. The use of such media andagents for pharmaceutically active substances is well known in the art.Preferably, the carrier is suitable for oral, intravenous,intramuscular, subcutaneous, parenteral, spinal or epidermaladministration (e.g., by injection or infusion). Depending on the routeof administration, the active compound may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

Conjugates of the invention can also be administered parenterally. Thephrase “administered parenterally” as used herein means modes ofadministration other than oral and topical administration, usually byinjection, and includes, without limitation, intravenous, intramuscular,intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. The active compounds can be prepared withcarriers that will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation.

One of ordinary skill in the art can determine and prescribe theeffective amount of the pharmaceutical composition required. Forexample, one could start doses of the known or novel photosensitizercomposition levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved.

Other Embodiments

The compositions of the invention can be used to decontaminate inanimateobjects, such as medical and dental devices, heat-sensitive filters,surfaces of transilluminators, sonication probes, and any other surfacethat is light accessible and carries unwanted organisms. Many of thesedevices cannot be autoclaved, in which case the methods and compositionsof the invention can be useful for decontamination.

The methods and compositions of the invention can be incorporated into akit, which contain one or more of a photosensitizer which may or may notbe coupled to a backbone, one or more target moieties, a coupling orcross-linking agent, buffers, and instructions for use. The user of thekit can select an appropriate target moiety to apply to the particularunwanted organism of choice. Two or more target moieties can be coupledto the photosensitizer-backbone, such that a broader range of unwantedorganisms can be eliminated or substantially reduced by application of asingle product.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting. The contents of allreferences, pending patent applications and published patents, citedthroughout this application, are hereby expressly incorporated byreference.

EXAMPLES Example 1 Preparation of polylysine-chlorin e6 Conjugates ofVarying Charges

The N-hydroxy succinimide (NHS) ester of the photosensitizer chlorin e6,was prepared by reacting 1.5 equivalents of dicyclohexylcarbodiimide and1.5 equivalents of NHS with one equivalent of chlorin e6, molecule I inFIG. 1A, or other photosensitizer in dry dimethylsulfoxide (DMSO) toform the NHS-ester, molecule II in FIG. 1A. The procedure described forthe material prepared for use in these Examples, with thephotosensitizer chlorin e6, is suitable also for preparation of estersof benzoporphyrin derivative, or any carboxyl containing tetrapyrrolephotosensitizer. After incubation in the dark at room temperature for 24h, the NHS ester was frozen in aliquots for further use. Poly-lysine(molecule III of FIG. 1B) hydrobromide (HBr, 50 mg) of either L or Doptical configuration, and of a range of molecular weights (molecularweight 40,000 to 60,000, equivalent to 22,000 to 33,000 polylysine freebase) was dissolved in 50 ml dry DMSO, and N-ethylmorpholine (1 ml) wasadded. To this solution was added dry DMSO (1 ml) containingphotosensitizer-NHS ester (25 mg). The solution was incubated in thedark at room temperature for 24 h, to synthesize the polylysine chlorine6 conjugate, molecule III in FIG. 1B, and also shown in FIG. 1C.

Charge modification was effected as follows.

To neutralize positive charges on the polylysine the molecules werereacted with an acylating agent, acetic anhydride. A sample ofpolylysine chlorin e6 conjugate (molecule III in FIG. 1D) solution wastreated with an excess of acetic anhydride (100 mg dissolved in 0.5 mldry DMSO) to produce an uncharged neutral acetic acid amide conjugate.The resulting molecule was uncharged. One can modify these conditions toproduce molecules of other charges. This allows one to make and testvarious possibilities and choose the best for application to aparticular conjugate or target organism.

To convert positive charges on the polylysine to negative charges, themolecules were reacted with an acylating agent, succinic anhydride. Asample of the polylysine chlorin e6 in DMSO was treated with an excessof succinic anhydride (100 mg dissolved in 0.5 ml dry DMSO) to producethe succinic acid amide, and convert the positively charged amino groupsto negatively charged carboxylic acid groups. The resulting molecule hadall lysines converted to negative charges. One can modify the conditionsto produce molecules of other charges. This would allow one to make andtest various possibilities and choose the best for application to aparticular conjugate or target organism.

After incubation of the conjugated molecules, and the acylatedcharge-modified conjugates, in the dark at room temperature for 24 h,the solutions were transferred to dialysis tubes having the correctmolecular weight cutoff to permit dialysis of polylysine, using dialysismaterial resistant to DMSO, and dialyzed for 24 h against 3 changes of10 mM phosphate buffer (pH 7). FIG. 1D shows molecule V, which forchlorin e6 polylysine acetic acid amide neutral conjugate the R groupindicates —COCH₃, and for chlorin e6 polylysine succinic acid amide theR group indicates —COCH₂CH₂COOH.

Example 2 Preparation of histatin-chlorin e6 Conjugates of VaryingCharges

Histatin-5 (10 mg) was dissolved in 2 ml of 0.1 M Na₂CO₃ buffer, pH 9.5,and was mixed with 0.1 ml DMSO containing 5 mg of chlorin e6N-hydroxysuccinic amide, prepared as described in Example 1. Thereaction was continued further by incubation for 24 h at roomtemperature in the dark, and the solution was dialyzed three timesagainst 10 liters of PBS. The resulting green precipitate was dissolvedin 2 ml 0.1 M Na₂CO₃ buffer, pH 10.5.

Measurements of the absorbance spectrum indicated that, assuming theextinction coefficient at 400 nm of chlorin e6 was unchanged bycomplexation, i.e., is 1.5×10⁵ M⁻¹, cm⁻¹, and that all chlorin e6remaining after dialysis was covalently attached to the histatin, it wasdetermined that 4 chlorin e6 molecules were attached per histatin-5peptide chain. Although histatin-5 is basic, the conjugate was found tobe acidic, since lysine amino groups were replaced with two carboxylgroups on the chlorin. The structure is shown in FIG. 1C.

The ratio of histatin to chlorin e6 can be varied by altering the ratioof the two species in the reaction.

Example 3 Preparation of histatin-polylysine-chlorin e6 Conjugates ofVarying Charges

The DMSO solution of polylysine-chlorin e6 referred to in Example 1 wastreated with a solution of N-succinimidyl-3-[2-pyridyldithiopropionate](SPDP) in DMSO to form polylysine chlorin e6-SPDP, Molecule VI in FIG.2A. The amount depends on the molecular weight of the polylysine butshould be about three to four equivalents per polymer chain. Thereaction is incubated for 24 h in the dark at room temperature. Thepolylysine-photosensitizer-SPDP solution is dialyzed as above.

The histatin (or other polypeptide containing at least one lysineresidue, 5 mg) is dissolved in 2 ml phosphate buffer (SATA, 50 mM, pH7.5 containing 1 mM EDTA) and treated with N-succinimidyl-S-thioacetate(0.5 mg dissolved in DMSO, 100 μl) for 1 h at room temperature as shownin reaction 6 of FIG. 2B, to form SATA-histatin, molecule IX in FIG. 2BThe solution of the peptide is then treated with 200 μl of a solutioncontaining hydroxylamine hydrochloride 0.5 mM, EDTA 25 mM and sodiumphosphate 50 mM, pH 7.5, and incubated for 2 h at room temperature toform the deprotected SATA derivative, molecule X in FIG. 2B. Thesolution is then desalted on a P4 column, and eluted with phosphatebuffer (50 mM, pH 7.5) containing 10 mM EDTA. This results in theproduction of a peptide containing free thiol group.

The peptide containing free thiol group derivative of polylysine chlorine6, molecule VII in FIG. 2A, which can be positively, neutrally ornegatively charged, is then reacted with the SPDP in the ratio of therelative molecular weight of peptide and polylysine, for 24 h at roomtemperature. The product comprising a peptide-polylysinechlorin e6conjugate shown in FIG. 2C is then purified on a Sephadex G50 column.

Example 4 Photosensitizer chlorin e6 Conjugate Uptake

Porphyromonas gingivalis 381, a Gram negative bacterium and one of themost common species in dental plaque, was maintained by weeklysubculture in Trypticase Soy Agar (TSA) with 1% hemin, 1% vitamin K and5% sheep blood. For experimental purposes, the organism was grownanaerobically in a chamber with 80% N₂, 10% CO₂ at 35° C. for 48 h,harvested by centrifugation and resuspended in Trypticase Soy Broth(TSB) with 1% hemin and 1% vitamin K. Cells were dispersed by sonicationand repeated passage through Pasteur pipettes. Cell numbers weremeasured in a spectrophotometer (at wavelength 600 nm, in which an O.D.of 1 yields 10⁸ cells/ml) using one ml tubes, to obtain the appropriatenumber of bacteria for each experiment (10⁹ cells/ml for uptake studiesand 10⁸ cells/ml for irradiation studies).

Hamster cheek pouch squamous cell carcinoma cell line (HCPC-1) cellswere used (Odukoya, O. et al., JNCI 71:6, 1253-1258, 1983). Cells weregrown in Dulbecco's modified Eagle's medium (DMEM) with high glucose(Gibco, Grand Island, N.Y.) supplemented with heat-inactivated 10% fetalcalf serum (FCS, Gibco), 100 units/ml penicillin G and 100 μg/mlstreptomycin (Sigma, St. Louis, Mo.). Medium was changed every 2-3 daysand cells were passaged weekly using trypsin-EDTA. All cells weremaintained in 10 cm-diameter culture dishes with 12 ml growth medium at37° C. in a humidified, 95% air, 5% CO₂ atmosphere.

The following photosensitizing conjugates of the invention ofpoly-1-lysine with chlorin e6 (M. Wt. 1,000 to 3,000) with positive,negative and neutral charges, were used: polylysine chlorin e6(cationic, unacylated); polylysine chlorin e6-succinylated (anionic,polylysine-chlorin e6-succ); and polylysine chlorin e6 acetylated(neutral, polylysine-chlorin e6-ac). Synthesis is described inExample 1. Unless indicated, all media, buffers, solutions, andglassware used for growth and maintenance of bacterial and animal cellswere sterile.

Samples of suspensions of P. gingivalis (10⁹ cells/ml) were incubated intriplicate in the dark at room temperature for one min withphotosensitizer at 1, 5 and 10 μm chlorin e6 equivalent (finalconcentration in TSB). Cell suspensions were centrifuged, thephotosensitizer-containing supernatants aspirated and bacteria werewashed once with 1 ml sterile PBS. Cells were resuspended in 1.5 ml 0.1M NaOH/1% sodium dodecyl sulfate (SDS), and incubated for at least 24 hto yield a homogenous solution.

Fluorescence of each cell extract was measured on a spectrofluorimeter(model FluoroMax, SPEX Industries, Edison, N.J.). The excitationwavelength was 400 nm and emission spectra of cell suspensions wererecorded from 580 to 700 nm. The protein content of each cell extractwas determined by a modified Lowry method (Markwell M. A. et al. Anal.Biochem. 87:206, 1978) using bovine serum albumin dissolved in 0.1 MNaOH/1% SDS as a protein standard to construct calibration curves.Results were expressed as mol chlorin e6 taken up per mg cell protein.

HCPC-1 cells in exponential growth phase were trypsinized and countedusing a hemocytometer. Each well of 24-well culture plates was seededwith 10⁵ cells in 1 ml growth medium containing 10% FCS, and wereincubated overnight to allow cells to attach and resume exponentialgrowth. Conjugates were added to wells in triplicate, as follows. Mediumwas removed and replaced with medium containing 10% FCS and theconjugates at concentrations indicated, and plates were incubated for 1min. The conjugate solutions were then aspirated from wells, cells werewashed once with 1 ml sterile PBS, and incubated with 1 ml trypsin-EDTAfor 10 min. The resulting cell suspensions were centrifuged, the trypsinsupernatant was aspirated and pellets were dissolved in 0.1 M NaOH/1%SDS. Fluorescence of each cell extract was determined as describedabove.

The uptake of conjugates as a function of concentration is shown inFIGS. 3 and 4, for P. gingivalis and HCPC-1 cells, respectively(ordinates in these Figures differ by a factor of 50). Uptake of chlorine6 conjugates was dose-dependent. Cells of P. gingivalis and HCPC-1cells accumulated 2 and 2 to 4 times more, respectively, of the cationicconjugate compared to the anionic and neutral conjugates, at eachconcentration. A high degree of selectivity of accumulation of thesephotosensitizer conjugates into bacteria, compared to that for mammaliancells, is observed.

Example 5 Phototoxicity of e6 Conjugates

In order to establish the effectiveness and selectivity of theseconjugates for killing bacteria while sparing mammalian cells, the threeconjugates were compared to two widely used clinical photosensitizers,Photofrin II and benzoporphyrin derivative (BPD) (QLT PhototherapeuticsInc., Vancouver, BC). Photosensitizer bulk solid was dissolved in DMSOat 1 mM and diluted in TSB.

Suspensions of P. gingivalis cells (10⁸/ml) were incubated in duplicatein the dark at room temperature for 1 min with 5 μm chlorin e6equivalent of each conjugate, with Photofrin II, and with BPD. Cellswere centrifuged, washed once with sterile PBS, and 1 ml fresh TSB wasadded. In the Example here, the ratios of uptake of chlorin e6 per mgcell protein in P. gingivalis and HCPC-1 cells for the cationic, anionicand neutral conjugates were 46:1, 60:1 and 22:1, respectively.

Bacterial suspensions were added to wells of 12-well plates, and wereirradiated in the dark at room temperature using a light emitting diodearray which emitted light with wavelengths from 630-710 nm, spanning theabsorbance maxima of these photosensitizer compositions. Wells wereexposed from below, using fluences from 0 to 25 J/cm² at an irradianceof 65 mW/cm². Plates were covered during illumination to maintainsterility of the cultures. After illumination of the appropriate wells,serial dilutions of the contents of each well were prepared in TSB, andduplicate 100 μl aliquots were spread on the surfaces of blood agarplates. Survival fractions in each well were calculated by counting thecolonies on the plates and dividing by number of colonies fromunirradiated controls incubated with photosensitizer in the dark at roomtemperature for periods equal to irradiation times. Other controls were:bacteria treated neither with photosensitizer nor light, and incubatedat room temperature in the dark, and cells exposed to light in theabsence of photosensitizer.

HCPC-1 cells, 2×10⁴ in aliquots of 100 μl growth medium with 10% FCS,were seeded in 96-well plates and cultured for 24 h until 70% confluent.Six wells from each plate were incubated with 5 μM of eachphotosensitizer and irradiated using the conditions described in Example3. After illumination, cells were incubated with fresh medium at 37° C.for 24 h. Control cells were incubated under the conditions as inExample 3. Cell viability was determined 24 h after irradiation usingthe MTT-microculture tetrazolium assay, a method that assessesdehydrogenase activity in mitochondria of live cells (Mosmann T. J.Immunol. Methods 65, 55-63, 1983), and survival fractions werecalculated as 570 nm absorbance of treated cells divided by that ofunirradiated controls.

FIGS. 5 and 6 show that the conjugates are highly selectively phototoxicto P. gingivalis compared to mammalian cells. The cationic conjugatepolylysine-chlorin e6 killed 99.9% of bacteria, and less than 2% ofHCPC-1 cells. High selectivity of the neutral conjugate,polylysine-chlorin e6-ac, which killed over 90% of bacteria and notHCPC-1 cells, is also shown. The anionic conjugate polylysine-chlorine6-succ caused a 66% reduction in viability of P. gingivalis, and HCPC-1cells were not killed by this photosensitizer.

In contrast, PFII and BPD did not show killing at the indicatedconcentrations, except for BPD which killed HCPC-1 cells and not P.gingivalis.

Example 6 In Vivo Studies in Animal Wound Models

Infected wounds are created on the dorsal skin of mice by using ascalpel to produce a 3 cm incision which is then inoculated with 10⁷ and10⁸ c.f.u. of a bacterial species. An infected bum is produced asdescribed by Stevens, E. J. et al., J. Burn. Care Rehabil. 15, 232-235,1994. Conjugates directed against either of the bacterial species orunconjugated chlorin e6 are injected either perilesionally orintravenously. The doses of conjugate, light, and the interval betweenthe injection and illumination are varied systematically. Responses totreatment are assessed by observing the rate of healing of the wound andthe burn. Tissue samples (2 mm punch biopsies) are taken at intervalsafter treatment to determine the quantity the bacteria, and to provideslides for histopathological evaluation. Bacterial colonization inwounds is quantitated by establishing c.f.u./g tissue, and by opticalmonitoring of the luciferase transfected P. aeruginosa bacterial strain.

OTHER EMBODIMENTS

Where a value for a physical parameter, e.g., molecular weight or numberof amino acid residues is given herein, the values can describe apopulation of molecules all or substantially all of which exhibit thatvalue or, to a population of molecules wherein the value represents anaverage, mean, or mode value for that parameter for the population. Thespecification of a physical parameter, e.g., the size of a peptide orprotein polymer, by number of residues or by molecular weight, caninclude the possibility of a degree of heterogeneity in the number ofresidues or in the molecular weight, such as occurs in the process ofchemical synthesis of such polymers and which can be reduced but notgenerally entirely eliminated by purification processes prior to furtheruse. Thus, for example, a preparation of polylysine indicated as beingcomprised of 10 lysine residues may consist of 80%, 90%, 95%, 99% or99.9% of the molecules being of this length, with the remaining 20%,10%, 5%, 1% or 0.1% of molecules having for example 9 or 8 residues, ormore rarely, 11 or 12 residues.

The conjugates described herein can be synthesized by substituting amixed population for one or more of the moieties discussed herein. Forexample, a pure preparation of a single photosensitizer moiety may beadded to a reaction mix containing a mixture of backbone or targetingmoiety substrates, to produce conjugates which include a single type ofphotosensitizer moiety conjugated to a mixture of targeting moieties, orto a mixture of backbone or targeting moiety chemical entities.

With regard to conjugates described herein a mixture may be constitutedin a formulation of the conjugate prior to use, for example, aformulation intended for application to a mixture of unwanted organismsmay be prepared as a mixture of two or more conjugates, each conjugatehaving an optimal affinity for one or more unwanted organisms, such thata plurality of target organisms can be reduced or eliminated.

The invention also includes fragments, preferably biologically activefragments, or analogs of a histatin, for example, histatin-5. Abiologically active fragment or analog is one having any in vivo or invitro activity which is characteristic of the histatin sequence.Particularly preferred fragments are fragments, e.g., active fragments,which are generated by de novo synthesis, proteolytic cleavage oralternative splicing events. Because peptides such as histatins oftenexhibit a range of physiological properties and because such propertiesmay be attributable to different portions of the molecule, a usefulhistatin fragment or histatin analog is one which exhibits a biologicalactivity in any biological assay for histatin activity. Most preferablythe fragment or analog possesses at least 20% of the activity of thefull-length naturally occurring histatin in any in vivo or in vitrohistatin assay.

Analogs can differ from naturally occurring histatin in amino acidsequence or in ways that do not involve sequence, or both. Non-sequencemodifications include in vivo or in vitro chemical derivatization ofhistatin. Non-sequence modifications include changes in acetylation,methylation, phosphorylation, carboxylation, or glycosylation.

Preferred analogs include histatin (or biologically active fragmentsthereof) whose sequences differ from the wild-type sequence by one, two,three, four, or five or more conservative amino acid substitutionsand/or by one, two, three, four, or five or more non-conservative aminoacid substitutions, deletions, or insertions which do not abolish thehistatin biological activity. Conservative substitutions typicallyinclude the substitution of one amino acid for another with similarcharacteristics, e.g., substitutions within the following groups:valine, glycine; glycine, alanine; valine, isoleucine, leucine; asparticacid, glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine. Other conservative substitutionscan be taken from the table below.

Other analogs within the invention are those with modifications whichincrease peptide stability; such analogs may contain, for example, oneor more non-peptide bonds (which replace the peptide bonds) in thepeptide sequence. Also included are: analogs that include residues otherthan naturally occurring L-amino acids, e.g., D-amino acids ornon-naturally occurring or synthetic amino acids, e.g., β or γ aminoacids; and cyclic analogs.

As used herein, the term “fragment”, as applied to a histatin analog,will ordinarily be of sufficient length to confer biological activity,e.g., at least 20% of the binding activity of a full length molecule. Inpreferred embodiments the fragments are 12 residues long or longer.Fragments of a histatin can be generated by methods described herein andby methods known to those skilled in the art. The ability of a candidatefragment to exhibit a biological activity of histatin can be assessed bymethods known to those skilled in the art and as described herein.

In order to obtain a histatin polypeptide, histatin-encoding DNA can beintroduced into an expression vector, the vector introduced into a cellsuitable for expression of the desired protein, and the peptiderecovered and purified, by prior art methods. Preferably histatinpeptides are produced in vivo as a fusion to a larger protein, and arecleaved into the fragment after initial purification from a cellextract. Preferably the histatin peptides are synthesized chemically,for example, on a peptide synthesizer.

TABLE 1 CONSERVATIVE AMINO ACID REPLACEMENTS For Amino Acid Code Replacewith any of Alanine A D-Ala, Gly, beta-Ala Arginine R D-Arg, Lys, D-Lys,homo-Arg, D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine ND-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic Acid D D-Asp, D-Asn,Asn, Glu, D-Glu, Gln, D-Gln Cysteine C none in parent therefore use isnot preferred Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-AspGlutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine G Ala,D-Ala, Pro, D-Pro, β-Ala Acp Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu,Leucine L D-Leu, Val, D-Val, Leu, D-Leu Lysine K D-Lys, Arg, D-Arg,homo-Arg, D-homo-Arg, Ile, D-Ile, Orn, D-Orn Methionine M none in parenttherefore use is not preferred Phenylalanine F D-Phe, Tyr, D-Thr,L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4, or 5-phenylproline, cis-3,4,or 5-phenylproline Proline P D-Pro, L-1-thioazolidine-4-carboxylic acid,D-or L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr,allo-Thr, phosphoser Threonine T D-Thr, Ser, D-Ser, phosphoser,allo-Thr, Val, D-Val Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, D-HisValine V D-Val, Leu, D-Leu, Ile, D-Ile

Preferred histatin-1 analogs are those in which:

R1 is asp or is deleted

R2 is ser or phosphoserine or is deleted

R3 is his or is deleted

R4 is ala or is deleted

R5 is lys or arg or glu or asp

R6 is arg or lys

R7 is his

R8 is his

R9 is gly or ala

R10 is tyr or phe

R11 is lys or arg

R12 is arg or lys

R13 is lys or arg

R14 is phe or tyr

R15 is his

R16 is glu or asp

R17 is lys or arg

R18 is his

R19 is his

R20 is ser or thr or phosphoser

R21 is his or is deleted

R22 is arg or lys or is deleted

R23 is gly or glu or asp or is deleted

R24 is tyr or phe or is deleted

R25 is pro or arg or lys or is deleted

R26 is phe or tyr or leu or ile is deleted

R27 is phe or tyr or leu or ile or thr or ser or is deleted

R28 is gly or ala or is deleted

R29 is asp or glu or is deleted

R30 is phe or tyr or leu or ile or is deleted

R31 is gly or ala or is deleted

R32 is deleted or ser or thr or phosphoser

R33 is deleted or asn or gln

R34 is deleted or tyr or phe

R35 is deleted or leu or ile or tyr or phe

R36 is tyr or phe or leu or ile or is deleted

R37 is deleted or asp or glu

R38 is deleted or asn or gin

Preferred histatin-3 analogs are those in which:

R1 is asp or is deleted

R2 is ser or phosphoserine or is deleted

R3 is his or is deleted

R4 is ala or is deleted

R5 is lys or arg or glu or asp

R6 is arg or lys

R7 is his

R8 is his

R9 is gly or ala

R10 is tyr or phe

R11 is lys or arg

R12 is arg or lys

R13 is lys or arg

R14 is phe or tyr

R15 is his

R16 is glu or asp

R17 is lys or arg

R18 is his

R19 is his

R20 is ser or thr or phosphoser

R21 is his or is deleted

R22 is arg or lys or is deleted

R23 is gly or glu or asp or is deleted

R24 is tyr or phe or is deleted

R25 is arg or lys or is deleted

R26 is deleted or ser or thr or phosphoser

R27 is deleted or asn or gln

R28 is deleted or tyr or phe

R29 is deleted or leu or ile or tyr or phe

R30 is tyr or phe or leu or ile or is deleted

R31 is deleted or asp or glu

R32 is deleted or asn or gin

Preferred histatin-5 analogs are those in which:

R1 is asp or is deleted

R2 is ser or phosphoserine or is deleted

R3 is his or is deleted

R4 is ala or is deleted

R5 is lys or arg or glu or asp

R6 is arg or lys

R7 is his

R8 is his

R9 is gly or ala

R10 is tyr or phe

R11 is lys or arg

R12 is arg or lys

R13 is lys or arg

R14 is phe or tyr

R15 is his

R16 is glu or asp

R17 is lys or arg

R18 is his

R19 is his

R20 is ser or thr or phosphoser

R21 is his or is deleted

R22 is arg or lys or is deleted

R23 is gly or glu or asp or is deleted

R24 is tyr or phe or is deleted

In preferred embodiments the targeting moiety includes a histatin, or anactive fragment or analog thereof, e.g., histatin-1 through -8,preferably histatin-1, -3, or -5. In preferred embodiments the targetingmoiety includes a fragment of a histatin, e.g., histatin-5. In preferredembodiments the targeting moiety includes histatin-5 residues 13-24, orcorresponding residues from other histatins. In preferred embodimentsthe targeting moiety includes a histatin molecule which has beenengineered to include an internal duplication.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, that the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications within the scope of the inventionwill be apparent to those skilled in the art to which the inventionpertains.

What is claimed is:
 1. A method of treating a subject for a disorder ofthe oral cavity characterized by the presence of an unwanted organismcomprising: administering to the subject, a conjugate comprising apolylysine backbone to which is coupled a histatin targeting moiety anda porphyrin photosensitizer; irradiating the subject with energy of awavelength appropriate to produce a cytotoxic effect by thephotosensitizer; thereby treating the subject, for the disordercharacterized by the presence of an unwanted organism.
 2. A method oftreating a subject for a disorder of the oral cavity characterized bythe presence of an unwanted organism comprising: administering to thesubject, a polycationic photosensitizer conjugate comprising chlorin e6coupled to polylysine; irradiating the subject with energy of awavelength appropriate to produce a cytotoxic effect by thephotosensitizer; thereby treating the subject, for the disordercharacterized by the presence of an unwanted organism.
 3. A method oftreating a subject for a disorder characterized by the presence of anunwanted organism comprising: administering to the subject, apolycationic photosensitizer conjugate comprising chlorin e6 coupled topolylysine; irradiating the subject with energy of a wavelengthappropriate to produce a cytotoxic effect by the photosensitizer;thereby treating the subject, for the disorder characterized by thepresence of an unwanted organism.
 4. A method of treating a subject fora disorder of the oral cavity characterized by the presence of anunwanted bacterium comprising: administering to the subject, apolycationic photosensitizer conjugate comprising chlorin e6 coupled topolylysine; irradiating the subject with energy of a wavelengthappropriate to produce a cytotoxic effect by the photosensitizer;thereby treating the subject, for the disorder characterized by thepresence of an unwanted organism.
 5. A method of treating a subject, fora disorder characterized by the presence of an unwanted bacterium,comprising: administering to the subject, a polycationic photosensitizerconjugate comprising chlorin e6 coupled to polylysine; providing thesubject with irradiation of energy of a wavelength appropriate toproduce a cytotoxic effect by the chlorin e6; thereby treating thesubject, for the disorder.